Genetically modified cells that produce c6-c10 fatty acid derivatives

ABSTRACT

Genes encoding mutant 3-ketoacyl-CoA synthases are introduced into host cells. Certain of the mutants enhance the production of shorter-chain fatty acids and derivatives by the cell than do the wild-type (unmutated) enzymes. In other cases, the chain length is not significantly affected, but productivity is enhanced. In specific cases, both a shift toward lower chain length and higher productivity is seen. Cells producing the mutant 3-ketoacyl-CoA synthases are especially suitable for producing C6-C10 fatty acids and derivatives.

This work was supported by the United States Department of Energy undercontract no. DE-EE0070007. The United States Government has certainrights to this invention.

This invention relates to genetically modified cells that produce C6-C10fatty acids and fatty acid derivatives such as C6-C10 fatty acid estersand C6-C10 fatty alcohols.

Fatty acids and fatty acid derivatives are useful as solvents forlacquers, paints, varnishes and other compositions; as plasticizers fororganic resins, as fragrances and flavorings, as fuels for jet and otherinternal combustion engines, and as raw materials for making a varietyof downstream products. Accordingly, the fatty acids and derivatives arecurrently produced industrially using non-renewable fossil fuels as thecarbon source, or from oils derived from plants such as palm or coconut.Selectivity towards a specific chain length is an important shortcomingin these processes—the products tend to be a mixture of compounds havinga range in number of carbon atoms. Separating the compounds intodiscrete chain lengths is difficult, and often much of the feedstock isconverted to lower-value products that do not have the requisite numberof carbon atoms.

Biological cells naturally produce fatty acid derivatives. For example,almost all living cells produce triglycerides of fatty acids as well asother fatty acid esters. The triglycerides and other esters playimportant roles in the metabolism, cellular structure, and otherbiological processes of the cells, and can perform other usefulfunctions such as storing energy. Biological processes potentially offera way to produce fatty acid derivatives industrially. Among otherpotential advantages, biologically-produced fatty acid and fatty acidderivative production in some cases can rely on annually renewablecarbon sources such as sugars, rather than on fossil fuels.

The fatty acid groups produced in biological systems tend to have chainlengths of 12 carbon atoms or greater. Thus, naturally-occuring cellsare a good source for C12 and higher fatty acids and derivatives. Forexample, triglycerides produced naturally by these cells can behydrolyzed to produce C12 or higher fatty acids, which can in turn beconverted to other derivatives such as esters or alcohols. On the otherhand, few cells naturally produce fatty acids of C6-C10 chain length insignificant quantities.

Shorter-chain fatty acids can be produced from biologically-producedoils and fat (and/or their constituent fatty acids), but doing sorequires further capital costs for the necessary processing equipmentand operating costs to effect the conversion to shorter chain lengths.In addition, the cost of feedstocks can vary considerably from year toyear or from one geographical location to another due to weather effectson crop production.

Biological cells produce fatty acid derivatives through a nativemetabolic pathway that starts with acetyl-CoA and malonyl-ACP.Acetyl-CoA condenses with malonyl-ACP with loss of carbon dioxide andCoA to produce 3-ketobutyryl-ACP. Subsequent enzymatic reactions convertthe 3-ketobutyryl-ACP successively to 3-hydroxybutyryl-ACP, then totrans-2-butenoyl-ACP (with loss of water) and finally to butyryl-ACP.The butyryl-ACP can re-enter this reaction cycle in place of acetyl-CoAto produce hexanoyl-ACP. This cycle repeats itself, producing in eachiteration a longer carbon atom chain by adding two carbon atoms at atime, until terminated by some other cellular process. However, thisnative ACP-dependent pathway has low termination rates for C6-C10 chainlengths, resulting in low production of C6-C10 fatty acids and fattyacid derivatives.

Okamura et al., in PNAS vol. 107, no. 25, pp. 11265-11270 (2010)reported that the enzyme produced by the nphT7 gene of a soil-isolatedStreptomyces strain catalyzes a single condensation of acetyl-CoA andmalonyl-CoA to produce acetoacetyl-CoA.

As further described in US 2014/0051136, C4 fatty acid derivatives canbe produced via a CoA-dependent pathway instead of the nativeACP-dependent pathway by a cell modified to include the nphT7 gene andfurther non-native genes that encode a 3-ketoacyl-CoA reductase, a3-hydroxyacyl-CoA dehydrase and a trans-2-enol-CoA reductase. However,the nphT7 gene is highly selective to the reaction of acetyl-CoA andmalonyl-CoA to produce C4 fatty acids and derivatives. Unlike the nativeACP-dependent pathway, the CoA-dependent pathway based on the nphT7 geneproduces few fatty acid derivatives having C6 or greater chain length.Thus, as shown in US 2014/0051136, cells modified in this manner produceonly small proportions of C6 or higher fatty acid derivatives.

WO 2015/10103 describes modifications to the NphT7 enzyme that permit itto more efficiently catalyze the condensation of longer-chain acyl-CoAcompounds with malonyl-CoA. Certain strains were modified to includeboth the wild-type nphT7 gene and a variant of that gene. These cellsproduce greater relative quantites of longer fatty acids, butselectivity is poor. C6-C10 fatty acid derivatives in particular aremade in only small amounts.

Therefore, there remains a desire for a process that produces fattyacids in good yields and with good selectivities to C6-C10 fatty acidsor derivatives.

Applicants have discovered that C6-C10 fatty acids or derivatives may beproduced by modifying a cell with mutant 3-ketoacyl-CoA synthase genesthat produce in the cell certain mutant 3-ketoacyl-CoA synthase enzymes.

Naturally occurring 3-ketoacyl-CoA synthase enzymes that include atleast one of sub-sequences SEQ ID NO. 87, SEQ ID NO. 105 or SEQ ID NO.106, or a sub-sequence closely homologous to either of these, whenheterologously expressed in a cell, tend to produce predominately C10 orhigher fatty acids or derivatives, often with significant fractions ofC12 or higher compounds. Applicants have found that by making specificmutations within these sub-sequences, the chain length of the fattyacids or derivatives is shifted downward, with C6-C10 chain lengthsbecoming predominant, and production of C12 and higher compoundsbecoming negligable. In particular cases, high selectivity toward C6and/or C8 chain lengths is seen.

Therefore, this invention is in one aspect a 3-ketoacyl-CoA synthasehaving an amino acid sequence characterized by including at least one ofa) a sub-sequence at least 80% identical to SEQ ID NO. 1, provided thatamino acid residue 8 is leucine, valine, isoleucine or methionine andamino acid residue 2 is leucine or methionine or a sub-sequence at least80% identical to SEQ ID NO. 160, provided that amino acid residue 8 iscysteine, leucine, valine, isoleucine or methionine and amino acidresidue 2 is leucine, threonine or methionine; b) a sub-sequence atleast 80% identical to SEQ ID NO. 2, provided that amino acid residue 6is isoleucine or methionine and c) a sub-sequence at least 80% identicalto SEQ ID NO. 3, provided that amino acid residue 6 is isoleucine,methionine, threonine, cysteine, valine, glutamine, phenylalanine,aspartic acid, asparagine or tyrosine. The invention in a further aspectis a genetically modified cell comprising a heterologous nucleic acidsequence encoding such a 3-ketoacyl-CoA synthase.

Applicants have further discovered that making specific mutations withincertain heterologously expressing 3-ketoacyl-CoA synthases can increaseproduction (e.g. increased titer) of fatty acids (and fatty acidderivatives) of C6 to C10 chain length. Thus in other aspects, theinvention is a 3-ketoacyl-CoA synthase having an amino acid sequencecharacterized by including one or more of SEQ ID NO. 50, SEQ ID NO. 56,SEQ ID NO. 60, SEQ ID NO. 65, SEQ ID NO. 69 and SEQ ID NO. 170, and agenetically modified cell comprising a heterologous nucleic acidsequence encoding such a 3-ketoacyl-CoA synthase.

The invention is in particular aspects a mutant 3-ketoacyl-CoA synthasehaving SEQ ID NO. 119, and a genetically modified cell comprising aheterologous nucleic acid sequence encoding such a mutant 3-ketoacyl-CoAsynthase. These mutant 3-ketaoacyl-CoA synthases have been found toimprove production of compounds having straight-chain alkyl groups andspecificity to C6-C10 chain lengths in such compounds. In particularembodiments, the mutant 3-ketoacyl-CoA synthase may have any of SEQ IDNO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18,SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO.23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ IDNO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQID NO. 33, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38,SEQ ID NO. 39, SEQ ID NO. 92, SEQ ID NO. 93, any one of SEQ ID NOs121-157 or any one of SEQ ID NOs. 172-204.

The invention in other aspects includes methods of using themicrooganisms of any of the foregoing aspects to make compounds havingstraight-chain alkyl groups. In particular aspects, the invention is amethod for making one or more compounds having a straight-chain alkylgroup, comprising culturing a genetically modified cell of the inventionin a fermentation medium and recovering the compound(s) having astraight-chain alkyl group from the fermentation medium.

The following Table 1 contains a summary of 3-ketoacyl-CoA synthasessequences and sub-sequences referenced herein. In Table 1, “Asch” refersto Acinetobacter schindleri CIP 107287; “Asch-2” refers to Acinetobacterschindleri NIPH 900; “Ajoh-2” refers to Acinetobacter johnsii SH046;“Alwo” refers to Acinetobacter lwoffii SH145; “ANIPH71” refers toAcinetobacter sp NIPH 713; “Asch homologues” refers to Asch, Asch-2,Ajoh-2, Alwo and ANIPH71; “Pstu” refers to Pseudomonas stutzeri ATCC17588 and “Aagr” refers to Alishewanella agri BL06.

TABLE 1 Summary of 3-ketoacyl-CoA synthase sequences and sub-sequencesFull 3-ketoacyl- Seq Mutant/ CoA sequence Aligns to: ID Wild or sub-Amino No. Type sequence Organism Acid Nos.  1 Mutant Sub-sequence Aschhomologues 177-185  2 Mutant Sub-sequence Pstu 181-190  3 MutantSub-sequence Aagr 181-190  4 Mutant Sub-sequence Asch homologues 150-156Pstu 151-157 Aagr 151-157  5 Mutant Sub-sequence Asch, Asch-2, Ajoh-2290-296 ANIPH71, Alwo 289-295  6 Mutant Sub-sequence Asch, Asch-2,Ajoh-2 326-331 ANIPH71, Alwo 325-330  7 Mutant Sub-sequence Aschhomologues 177-185  8 Wild- Full sequence Asch  1-370 Type  9-21 MutantFull sequence Asch  1-370  22-25 Mutant Sub-sequence Asch-2  1-370 26-29 Mutant Full sequence Alwo  1-369  30 Mutant Full sequence Ajoh-2 1-370  31-34 Mutant Full sequence ANIPH71  1-369  35 Wild-type Fullsequence Ajoh-2  1-370  36 Mutant Full sequence Asch  1-370  37-39Mutant Full sequence Ajoh-2  1-370  40 Mutant Full sequence Pstu  1-373 41 Mutant Sub-sequence Aagr 181-190  42-43 Mutant Full sequence Aagr 1-372  44 Mutant Sub-sequence Asch homologues 150-185  45 MutantSub-sequence Asch homologues 150-238  46 Mutant Sub-sequence Aschhomologues 290-338  47 Mutant Sub-sequence Asch homologues 150-338  48Mutant Sub-sequence Asch homologues  1-149  49 Mutant Sub-sequence Aschhomologues  1-338  50 Mutant Sub-sequence Asch, Asch-2, Ajoh-2 275-281ANIPH71, Alwo 274-280  51 Mutant Sub-sequence Asch homologues 177-185 52 Mutant Sub-sequence Asch homologues 150-185  53 Mutant Sub-sequenceAsch homologues 150-238  54 Mutant Sub-sequence Asch homologues 150-338 55 Mutant Sub-sequence Asch homologues  1-338  56 Mutant Sub-sequenceAsch, Asch-2 291-300  57 Mutant Sub-sequence Asch, Asch-2 290-338  58Mutant Sub-sequence Asch, Asch-2 150-338  59 Mutant Sub-sequence Asch,Asch-2  1-338  60 Mutant Sub-sequence Asch, Asch-2, Ajoh-2 177-190  61Mutant Sub-sequence Asch, Asch-2, Ajoh-2 150-190  62 Mutant Sub-sequenceAsch, Asch-2, Ajoh-2 150-238  63 Mutant Sub-sequence Asch, Asch-2,Ajoh-2 150-338  64 Mutant Sub-sequence Asch, Asch-2, Ajoh-2  1-338  65Mutant Sub-sequence Asch, Asch-2, Ajoh-2 316-331 ANIPH71, Alwo 315-330 66 Mutant Sub-sequence Asch, Asch-2, Ajoh-2 290-338 ANIPH71, Alwo289-337  67 Mutant Sub-sequence Asch, Asch-2, Ajoh-2 150-338 ANIPH71,Alwo 150-337  68 Mutant Sub-sequence Asch, Asch-2, Ajoh-2  1-338ANIPH71, Alwo  1-337  69 Mutant Sub-sequence Asch, Asch-2, Ajoh-2266-274 ANIPH71, Alwo 265-273  70 Mutant Sub-sequence Asch, Asch-2,Ajoh-2 150-338 ANIPH71, Alwo 150-337  71 Mutant Sub-sequence Asch,Asch-2, Ajoh-2  1-338 ANIPH71, Alwo  1-337  72-81 Mutant Full sequenceAsch  1-370  86 Wild-type Full sequence Asch-2  1-370  87 MutantSub-sequence Asch homologues 177-185  88 Wild-type Full sequence Alwo 1-369  89 Wild-type Full sequence ANIPH71  1-369  90 Wild-type Fullsequence Pstu  1-373  91 Wild-type Full sequence Aagr  1-372  92-93Mutant Full sequence Asch  1-370  97 Mutant Sub-sequence Asch, Asch-2,Ajoh-2 316-331 ANIPH71, Alwo 315-330 105 Mutant Sub-sequence Pstu181-190 106 Mutant Sub-sequence Aagr 181-190 107 Mutant Sub-sequenceAsch, Asch-2, Ajoh-2 266-274 ANIPH71, Alwo 265-273 108 MutantSub-sequence Asch homologues 275-281 109 Mutant Sub-sequence Asch,Asch-2 291-300 110- Mutant Full Sequence Asch homologues  1-370 111 112Mutant Full Sequence Asch, Asch-2  1-370 113 Mutant Full Sequence Asch,Asch-2, Ajoh-2  1-370 114- Mutant Full Sequence Asch, Asch-2, Ajoh-2 1-370 115 ANIPH71, Alwo  1-369 116 Mutant Sub-sequence Asch, Asch-2177-190 119- Mutant Full Sequence Asch homologues  1-370 159 160 MutantSub-sequence Asch homologues 177-185 161 Mutant Sub-sequence Aschhomologues 150-156 162 Mutant Sub-sequence Asch, Asch-2, Ajoh-2 326-331ANIPH71, Alwo 325-330 163 Mutant Sub-sequence Asch homologues 177-185164 Mutant Sub-sequence Asch homologues 150-185 165 Mutant Sub-sequenceAsch homologues 150-238 166 Mutant Sub-sequence Asch homologues 290-338167 Mutant Sub-sequence Asch homologues 150-338 168 Mutant Sub-sequenceAsch homologues  1-149 169 Mutant Sub-sequence Asch homologues  1-338170 Mutant Sub-sequence Asch homologues  51-59 171 Wild-typeSub-sequence Asch  51-59 172- Mutant Full Sequence Asch  1-370 204

Amino acid residues in all amino acid sequences described herein areordered in the N-terminus to C-terminus direction. “Upstream” means inthe direction toward the N-terminus, and “downstream” means toward theC-terminus direction. The “start” of an amino acid sequence is the firstamino acid residue in the N-terminus direction. The first amino acidresidue (amino acid residue 1) for any sequence or sub-sequencedescribed herein is the amino acid residue at its N-terminus.

A “sub-sequence” is a sequence of amino acid residues contained within alarger amino acid sequence.

“Identity” is used herein to indicate the extent to which two(nucleotide or amino acid) sequences have the same residues at the samepositions in an alignment. The identity is expressed herein as a %identity as determined using BLAST (National Center for BiologicalInformation (NCBI) Basic Local Alignment Search Tool) version 2.2.31software, using default parameters unless indicated otherwise in thisparagraph. Identity between amino acid sequences is determined usingprotein BLAST with the following parameters: Max target sequences: 100;Short queries: Automatically adjust parameters for short inputsequences; Expect threshold: 10; Word size: 6; Max matches in a queryrange: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1);Compositional adjustments: Conditional compositional score matrixadjustment; Filter: none selected; Mask: none selected. Nucleic acid %sequence identity between nucleic acid sequences is determined usingstandard nucleotide BLAST with the following default parameters: Maxtarget sequences: 100; Short queries: Automatically adjust parametersfor short input sequences; Expect threshold: 10; Word size: 28; Maxmatches in a query range: 0; Match/Mismatch Scores: 1, −2; Gap costs:Linear; Filter: Low complexity regions; Mask: Mask for lookup tableonly. A sequence having a % identity score of XX % (for example, 80%) toa reference sequence as determined in this manner is considered forpurposes of this invention to be XX % identical to or, equivalently,have XX % sequence identity to, the reference sequence.

For purposes of this invention, an amino acid residue of a sequence orsub-sequence under investigation “aligns” to an amino acid residue of areference sequence or sub-sequence when:

i) in the case of an entire sequence, the sequences are aligned usingthe BLAST version 2.2.31 software in the manner described above, and theamino acid residue of the sequence under investigation occupies the sameposition in the alignment as does the amino acid residue of thereference sequence;

ii) in the case of a sub-sequence, the sequence containing thesub-sequence under investigation is aligned with the reference sequence,and the amino acid residue of the sub-sequence under investigationoccupies the same position in the alignment as does the amino acidresidue of the reference sub-sequence.

For example, the 3-ketoacyl-CoA synthase enzyme of wild typeAcinetobacter schindleri CIP 107287 (SEQ ID NO. 8) includes thefollowing 9 amino acid residue sub-sequence.

Amino Acid Residue L N L S E V D A D Position in Sub-Sequence 1 2 3 4 56 7 8 9 Position in Entire 239 240 241 242 243 244 245 246 247 SequenceWhen the 3-ketoacyl-CoA synthase enzyme of wild type Acinetobacter spNIPH 713 (SEQ. ID. NO. 89) is aligned with SEQ ID NO. 8 using the BLASTsoftware as described, the following amino acid residues occupy the samepositions in the aligment:

A. schindleri sub-sequence L N L S E V D A D Position in A. schindleri 12 3 4 5 6 7 8 9 sub-sequence Position in entire A. 239 240 241 242 243244 245 246 247 schindleri sequence Acinetobacter sp. NIPH L N T S E — NA D 713 sub-sequenceThe leucine (L) of the Acinetobacter sp. NIPH sub-sequence aligns to theleucine at amino acid residue position 1 of the A. schindlerisub-sequence and also to the leucine at position 239 of the A.schlindleri 3-ketoacyl-CoA synthase sequence. A missing amino acidresidue of the Acinetobacter sp. NIPH sub-sequence aligns to the valine(V) at amino acid residue position 6 of the A. schindleri sub-sequenceand also to the valine of amine position 244 of the A. schlindleri3-ketoacyl-CoA synthase sequence. The second asparagine (N) residue ofthe Acinetobacter sp. NIPH sub-sequence aligns to the aspartic acidresidue at position 7 the of the A. schindleri sub-sequence and also tothe aspartic acid residue at position 245 of the A. schlindleri3-ketoacyl-CoA synthase sequence.

For purposes of this application, genetic material such as genes,promoters and terminators is “heterologous” if it is (i) non-native tothe host cell and/or (ii) is native to the cell, but is present at alocation different than where that genetic material is present in thewild-type host cell and/or (iii) is under the regulatory control of anon-native promoter and/or non-native terminator. Extra copies of nativegenetic material are considered as “heterologous” for purposes of thisinvention, even if such extra copies are present at the same locus asthat genetic material is present in the wild-type host strain.

An enzyme (such as a 3-ketoacyl-CoA synthase) is “heterologous” if it isnot produced by the wild-type host cell.

A “3-ketoacyl-CoA synthase” is an enzyme that catalyzes the condensationreaction of an acyl-CoA with malonyl-CoA to form a 3-ketoacyl-CoA. Theability of an enzyme to catalyze this reaction can be evaluated bymeasuring the release of free CoA-SH using5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent, malonyl-CoA as thedonor substrate, and a C4-C8 acyl-CoA as the primer substrate, in thepresence of the enzyme. The formation of the corresponding3-ketoacyl-CoA from any of these primer substrates indicates the enzymeis a 3-ketoacyl-CoA synthase.

The ability of an enzyme to catalyze this reaction can also be evaluatedusing butyryl-CoA as the primer substrate and malonyl-CoA as the donorsubstrate in the presence of 5 mM Mg⁺⁺ salt by measuring the increase inabsorbence at 303 nm as a function of the increase in the formation ofthe Mg⁺⁺-complex with the 3-ketohexanoyl-CoA product. 3-ketoacyl-CoAsynthase will produce 3-ketohexanoyl-CoA from butyryl-CoA primer,3-ketooctanoyl-CoA from haxanoyl-CoA primer, and 3-ketodecanoyl-CoA fromoctanoyl-CoA primer.

EC numbers are established by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB)(Enzyme Nomenclature 1992 [Academic Press, San Diego, Calif., ISBN0-12-227164-5 (hardback), 0-12-227165-3 (paperback)] with Supplement 1(1993), Supplement 2 (1994), Supplement 3 (1995), Supplement 4 (1997)and Supplement 5 (in Eur. J. Biochem. 1994, 223, 1-5; Eur. J. Biochem.1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997,250; 1-6, and Eur. J. Biochem. 1999, 264, 610-650). The EC numbersreferenced herein are derived from the KEGG Ligand database, maintainedby the Kyoto Encyclopedia of Genes and Genomics, sponsored in part bythe University of Tokyo. Unless otherwise indicated, the EC numbers areas provided in the database as of April 2009.

A “fatty acid” for purposes of this invention is a straight-chainmonoalkanoic acid having at least four carbon atoms.

A “derivative” of a fatty acid is a compound having a straight chainalkyl group formed in a series of one or more reactions at the site ofthe terminal carboxyl group of a fatty acid (or corresponding -CoAcompound), to convert the terminal carboxyl group (without loss of thecarboxyl carbon) to a different end group such as, for example, anester, an alcohol group, an amino group, an aldehyde group, a ketone, amethyl group, or an alkenyl group. The length of the straight-chainalkyl group of the fatty acid is preserved in any such derivative, andmay in some cases be extended.

A fatty acid ester is an ester compound corresponding to the reactionproduct of a fatty acid or a fatty acyl-CoA and an alcohol (with loss ofwater).

Chain lengths of fatty acids and derivatives are sometimes indicatedherein by the shorthand “CX”, wherein X is a number designating thenumber of carbon atoms. The number of carbon atoms designated in eachcase represents the carbon length of the straight-chain compound (afterremoval of CoA or ACP coenzymes) formed by the cell of the inventionthrough one or more iterations of the reaction cycle:

acyl-CoA (or acyl-ACP)+malonyl-CoA to form a 3-ketoacyl compound;

reduction of the 3-ketoacyl compounds to form a 3-hydroxyacyl compound;

dehydration of the 3-hydroxyacyl-CoA to form a 3-enoylacyl compound; and

reduction of the 3-enoylacyl compound to the corresponding acylcompound.

Each iteration of this reaction cycle adds two carbon atoms to thestarting acyl-CoA or acyl-ACP. The number of carbon atoms does notinclude additional carbon atoms that may be added during the formationof any derivatives of the fatty acid, such as, for example, carbonsincluded in an ester group. Thus, hexanoic acid methyl ester isconsidered as a “C6” fatty acid ester compound for purposes of thisinvention, the carbon of the methyl ester group not being counted.

In some embodiments, the genetically modified cell is a prokaryoticcell. In some embodiments, the genetically modified cell is a eukaryoticcell.

In some embodiments, the genetically modified cell is a microorganism,and may be a single-celled microorganism.

The host cell may be a plant cell, including a cell from a plant withinany of the Chlorophyta, Charophyta, Marchantiophyta, Anthocerotophyta,Bryophyta, Lycopodiophyta, Pteridophyta, Cycadophyta, Ginkgophyta,Pinophyta, Gnetophyta or Magnoliophyta plants. Such a plant cell may be,for example, a cell from a plant within any of the genera Arabidopsis,Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus,Saccharum, Salix, Simmondsia, and Zea.

The host cell may be a fungi, microalgae, algae or red algae(heterokont) cell. The host cell may be a yeast cell. A yeast or funguscell may be an oleaginous yeast or fungus, and/or may be a Crabtreenegative yeast or fungus.

The term “oleaginous fungi” refers to yeasts or filamentous fungi, whichaccumulate at least 10%, 12.5%, 15%, 17.5%, preferably at least 20% oreven at least 25% (w/w) of their biomass as lipid. They may evenaccumulate at least 30%, 40%, 50%, 60%, 70%, 80% (w/w) or more of theirbiomass as lipids. The biomass is usually measured as cell dry weight(CDW).

A “Crabtree-positive” organism is one that is capable of producingethanol in the presence of oxygen, whereas a “Crabtree-negative”organism is not. A yeast cell having a Crabtree-negative phenotype isany yeast cell that does not exhibit the Crabtree effect. The term“Crabtree-negative” refers to both naturally occurring and geneticallymodified organisms. Briefly, the Crabtree effect is defined as theinhibition of oxygen consumption by a microorganism when cultured underaerobic conditions due to the presence of a high concentration ofglucose (e.g., 10 g-glucose L-1). In other words, a yeast cell having aCrabtree-positive phenotype continues to ferment irrespective of oxygenavailability due to the presence of glucose, while a yeast cell having aCrabtree-negative phenotype does not exhibit glucose mediated inhibitionof oxygen consumption. Crabtree-positive yeast produce an excess ofalcohol rather than biomass production.

Examples of suitable yeast cells include, Pichia, Candida, Klebsiella,Hansenula, Kluyveromyces, Trichosporon, Brettanomyces, Pachysolen,Issatchenkia, Yamadazyma Saccharomyces, Schizosaccharomyces,Zygosaccharomyces, Debaryomyces, Cryptoococcus, Rhodotorula,Rhodosporidium, Lipomyces and Yarrowia. Examples of specific host yeastcells include C. sonorensis, K. marxianus, K. thermotolerans, C.methanesorbosa, Saccharomyces bulderi (S. bulderi), I. orientalis, C.lambica, C. sorboxylosa, C. zemplinina, C. geochares, P.membranifaciens, Z. kombuchaensis, C. sorbosivorans, C. vanderwaltii, C.sorbophila, Z. bisporus, Z. lentus, Saccharomyces bayanus (S. bayanus),D. castellii, C, boidinii, C. etchellsii, K. lactis, P. jadinii, P.anomala, Saccharomyces cerevisiae (S. cerevisiae) Pichia galeiformis,Pichia sp. YB-4149 (NRRL designation), Candida ethanolica, P.deserticola, P. membranifaciens, P. fermentans, Rhodosporidiumtoruloide, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C.pulcherrima, C. tropicalis, C. utilis, Trichosporon pullas, T. cutaneum,Rhodotorula glutinous, R. garminis, Yarrowia lipolytica andSaccharomycopsis crataegensis (S. crataegensis). Suitable strains of K.marxianus and C. sonorensis include those described in WO 00/71738 A1,WO 02/42471 A2, WO 03/049525 A2, WO 03/102152 A2 and WO 03/102201A2.Suitable strains of I. orientalis are ATCC strain 32196 and ATCC strainPTA-6648.

In some embodiments, the host cell is a bacteria cell. The bacteria maybe a gram-positive or gram-negative bacteria. It may be a cell withinany of the Chlamydiae, green nonsulfur, actinobacteria, planctomycetes,spirochaetes, fusobacteria, cyanobacteria, thermophilicsulphate-reducer, acidobacteria or proteobacteria classifications ofbacteria (Ciccarelli et al., Science 311 (5765): 1283-7 (2006).

Examples of suitable bacteria cells include, for example, those withinany of the genera Clostridium, Zymomonas, Escherichia, Salmonella,Rhodococcus, Pseudomonas, Streptomyces, Bacillus, Lactobacillus,Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,Corynebacterium, Bacteriophage, Brevibacterium, Acanthoceras,Acanthococcus, Acarvochloris, Achnanthes, Achnanthidiun, Actinastrum,Actinochloris, Actinocyclus, Actinotaenium, Amphichrsis, Amphidiniunm,Amphikrikos, Amplhipleura, Amphiprora, Amphithrix, Amphora, Anabaena,Anabaenopsis, Aneumnastus, Ankistrodesmius, Ankyra, Anomoeoneis,Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece,Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris,Asterionella, Asterococcus, Audouinella, Aularoseira, Bacillaria,Balbiania, Bambiusina, Bangia, Basichlamys, Batrarhospermum,Binurlearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus,Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia,Bulbochaete, Bumnilleria, Buinilleriopsis, Caloneis, Calothrix,Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Cenritractus,Centroniella, Ceratiunt, Chaetoceros, Chaetochloris, Chaetomorpha,Chaetonella, Chaetonemna, Chaetopeltis, Chaetophora, Chaetosphaeridium,Chamaesiphion, Chara, Characiochloris, Characiopsis, Characium,Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa,Chlamydomonas, Chlamydomonopsis, Chlamydomnyxa, Chlamydonephris,Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis,Chlorochytrium, Chloroccun, Chlorogloea, Chlorogloeopsis, Chlorogonium,Chlorolobion, Chloromonas, Chlorophysema, Cholorphyta, Cholorosaccus,Cholorosarcina, Choricystis, Chromophyton, Chromulina,Chroococcidiopsis, Chrococcus, Chroodactylon, Chroomonas, Chroothece,Chrysamoeba, Chysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella,Chrysochaete, Chrysohromulina, Chrysococcus, Chrysocrinus,Chrynsolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis,Chrysosaccus, Chrysophaerella, Chrysotephanosphaera, Clodophora,Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis,Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis,Compsopogon, Conjugatophyta, Conoehaete, Coronastrum, Cosmarium,Cosmnioneis, Cosmocladium, Crateriportula, Craticula, Crinalium,Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta,Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella,Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca,Cymatopleura, Cymbella, Cymbeilonitzschia, Cystodinium Dactylococcopsis,Debarya, Denticula, Dermatochrysis, Dermorarpa, Dermocarpella,Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon,Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula,Dichothrix, Dichtotomococcrus, Dicranochaete, Dictyochloris,Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia,Dilabifilum, Dimorphoccus, Dinobryon, Dinocuccus, Diplochloris,Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia,Dunaliella, Dysmorphaocuccus, Ecballocystis, Elakatothrix, Ellerbeckia,Encyonema, Enteromorpha, Entocladia, Entomoeis, Entophysalis,Ephichrysis, Epipyxis, Epithemia, Eremosphaura, Euastropsis, Euatstrum,Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia,Eustigmatophyta, Eutreptia, Fallcia, Ficherella, Fragilaria,Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia,Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeomonas,Gloeoplax, Gloeothece, Geloeotila, Gloeotrichia, Gloiodictyon,Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema,Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris,Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia,Gymnodiunium, Gymnozyga, Gyrosignma, Haematocuccus, Hafniomonas,Hallassia, Hammatoidea, Hannaea, Hantzchia, Hapalosiphon, Haplotaenium,Haptophyta, Haslea, Hemidinuim, Hemitonia, Heribaudiella, Heteromastix,Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix,Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus,Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum,Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas,Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia,Kathablepharis, Katodinium, Kaphyrion, Keratococcus, Kirchneriella,Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella,Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira,Lobococcus, Lobocystis, Lobomonas, Luticola, Lynbya, Malleochloris,Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocloleus,Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium,Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis,Microglena, Micromonas, Microspora, Microthamnion, Mischococcus,Monocrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia,Mougeotiopsis, Myochloris, Myromecia, Myxocarcina, Naegeliella,Nannochloris, Nautoccus, Navicula, Neglectella, Neidium, Nephroclamys,Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis,Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora,Onychonema, Oocadrium, Oocrystis, Opephora, Ophiocytium, Orthoseira,Oscillartoria, Oxyneis, Pachycladella, Palmella, Palmodictyon,Pnadorina, Pannus, Paralia, Pascherina, Paulshulzia, Pediastrum,Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema,Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus,Phaeaster, Phaeodermatium, Phaeophyta, Phaeoshaera, Phaeothamnion,Phormidium, Phycopeltis, Phyllariochloris, Phyllocadium, Phyllomitas,Pinnilaria, Pitophora, Placoneis, Planctonema, Planktophaeria,Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa,Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium,Pocillomanas, Podohedea, Polyblepharides, Polychaetophora, Polyedriella,Polyedriopsis, Polygoniochloris, Polyepidomanas, Polytaenia, Polytoma,Polytomella, Porphyridium, Posteriochromonas, Prasinochloris,Prasinocladus, Prasinophyta, Praisola, Prochlorphyta, Psammodictyon,Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria,Pseudochate, Pseaudocharacium, Pseudococcomyxa, Pseudodictyosphaerium,Pseudokephyrion, Pseudoncobrysa, Pseudoquadrigula, Pseudophaerocystis,Pseudostaurastrum, Pseudostraurosira, Pyrrophyta, Quadrichloris,Quadricoccus, Quadrigula, Radiocuccus, Radiobetalum, Raphidiopsis,Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhadorderma,Rhabomonas, Rhizoclonium, Rhodomonas, Rhodiphyta, Rhoicosenia,Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus,Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix,Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia,Scytonema, Slenastrum, Selenochloris, Sellaphora, Semiorbis,Siderocelis, Diderocys tops is, Dimonsenia, Siphononema, Sirocladium,Sirogonium, Skeletonema, Sorastrum, Spermatozopis, Sphaerellocystis,Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma,Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum,Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus,Stauroneis, Staurosira, Staurrosiella, Stenopterobia, Stephanocostis,Stephanodiscus, Stephanoporos, Stephanoshaera, Stichoccus, Stichogloea,Sigeoclonium, Stigonema, Stipitocuccus, Stokesiella, Stombomonas,Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella,Sykidion, Symploca, Synechococcus, Synechocystis, Synedra,Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum,Tetmemorus, Tetrachlorella, Tetracyclus, Tetrademus, Tetraedriella,tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira,Thanmiochaete, Thoakochloris, Thorea, Tolypella, Tolypothrix,Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria,Tribonema, Trichodesmium, Tricodiscus, Trochiscia, Tryblionella,Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria,Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium,Xanthophyta, Exencoccus, Zygenema, Zygnemopsis, and Zygonium.

Specific examples of bacteria host cells include Escherichia coli;Oligotropha carboxidovorans, Pseudomononas sp. Alcaligenes eutrophus(Cupriavidus necator), Bacillus licheniformis, Paenibacillus macerans,Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum,Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis,Bacillus subtilis, Cupriavidus basilensis, Cupriavidus campinensis,Cupriavidus gilardi, Cupriavidus laharsis, Cupriavidus metallidurans,Cupriavidus oxalaticus, Cupriavidus pauculus, Cupriaviduspinatubonensis, Cupriavidus respiraculi, Cupriavidus taiwanensis,Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium avium,Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium leprae,Mycobacterium marinum, and Mycobacterium ulcerans. In other embodiments,the bacterium is Nocardia sp. NRRL 5646, Nocardia farcinica,Streptomyces griseus, Salinispora arenicola, or Clavibactermichiganenesis.

The host cell may be a synthetic cell or a cell produced by a syntheticgenome, as described in U.S. Patent Publication 2007/0264688, or2007/0269862. The host cell may be a CHO cell, a COS cell, a VERO cell,a BHK cell, a HeLa cell, a Cvl cell, an MDCK cell, a 293 cell, a 3T3cell, or a PC12 cell.

In certain embodiments, the 3-ketoacyl-CoA synthase includessub-sequence SEQ ID NO. 1 or SEQ ID NO. 160, or a sub-sequence at least80% identical to SEQ ID NO. 1 or SEQ ID NO. 160, provided that that anamino acid residue that aligns to amino acid residue 8 in each case isany one of leucine, valine, isoleucine or methionine and an amino acidresidue that aligns to amino acid residue 8 is leucine or methionine inthe case of SEQ ID NO. 1 and leucine, methionine or threonine in thecase of SEQ ID NO. 160. SEQ ID NO. 1 differs from SEQ ID NO. 87 in thatthe threonine in amino acid residue position 8 of SEQ ID NO. 87 isreplaced in SEQ ID NO. 1 with any one of leucine, valine, isoleucine ormethionine. SEQ ID NO. 160 differs from SEQ ID NO. 87 in that thethreonine in amino acid residue position 8 of SEQ ID NO. 87 is replacedin SEQ ID NO. 160 with any one of leucine, valine, isoleucine ormethionine, and amino acid residue in position 2 may also be threonine.SEQ ID NO. 87 represents a sub-sequence found in a range ofnaturally-occuring 3-ketoacyl-CoA synthases. SEQ ID NO. 87 appears, forexample in the naturally-occuring 3-ketoacyl-CoA synthases ofAcinetobacter schindleri CIP 107287 (as amino acid residues 177-185 ofSEQ ID NO. 8, amino acid residue 178 being methionine); Acinetobacterschindleri NIPH 900 (as amino acid residues 177-185 of SEQ ID NO. 86,amino acid residue 178 being methioine); Acinetobacter johnsii SH046 (asamino acid residues 177-185 of SEQ ID NO. 35, amino acid residue 178being leucine); Acinetobacter lwoffii SH145 (as amino acid residues177-185 of SEQ ID NO. 88, amino acid residue 178 being leucine);Acinetobacter sp NIPH 713 (as amino acid residues 177-185 of SEQ ID NO.89, amino acid residue 178 being leucine). This substitution of thethreonine indicated at amino acid residue 8 of sub-sequence SEQ ID NO. 1or SEQ ID NO. 160 has been found to shift fatty acid production (andproduction of fatty acid derivatives) toward C6, C8 and/or C10 fattyacids and derivatives.

The 3-ketoacyl-CoA synthase in some embodiments includes at least onesub-sequence selected from a) SEQ ID NO. 4 or SEQ ID NO. 161, b) SEQ IDNO. 5 and c) SEQ ID NO. 6 or SEQ ID NO. 162. The 3-ketoacyl-CoA synthasepreferably includes each of a), b) and c).

In some embodiments in which SEQ ID NO. 4 or SEQ ID NO. 161 is presentin the 3-ketoacyl-CoA synthase, sub-sequence SEQ ID NO. 4 is presentupstream of subsequence SEQ ID NO. 1 or SEQ ID NO. 160, as the case maybe. The start of sub-sequence SEQ ID NO. 4 or SEQ ID NO. 161, as thecase may be, may be located, for example, 25 to 30 amino acid residues,especially exactly 27 amino acid residues, upstream of the start ofsub-sequence SEQ ID NO. 1 or SEQ ID NO. 160 (as the case may be) in such3-ketoacyl-CoA synthase enzymes.

The 3-ketoacyl-CoA synthase in some embodiments includes sub-sequenceSEQ ID NO. 44 and/or SEQ ID No. 45, or a sub-sequence at least 85%identical to either of those. As can be seen by comparing the sequences,each of SEQ ID NOs. 44 and 45 includes SEQ ID NO. 4 (which appears asamino acid residues 1-7 of SEQ ID NOs. 44 and 45) and SEQ ID NO. 1(which appears as amino acid residues 28-36 of SEQ ID NOs. 44 and 45).In sub-sequence SEQ ID NOs. 44 and 45, the start of sub-sequence SEQ IDNO. 4 is positioned 27 amino acid residues upstream of the start ofsub-sequence SEQ ID NO. 1.

The 3-ketoacyl-CoA synthase in some embodiments includes sub-sequenceSEQ ID NO. 164 and/or SEQ ID No. 165, or a sub-sequence at least 85%identical to either of those. As can be seen by comparing the sequences,each of SEQ ID NOs. 164 and 165 includes SEQ ID NO. 161 (which appearsas amino acid residues 1-7 of SEQ ID NOs. 164 and 165) and SEQ ID NO.160 (which appears as amino acid residues 28-36 of SEQ ID NOs. 164 and165). In sub-sequence SEQ ID NOs. 164 and 165, the start of sub-sequenceSEQ ID NO. 161 is positioned 27 amino acid residues upstream of thestart of sub-sequence SEQ ID NO. 160.

In some embodiments in which SEQ ID NO. 5 is present in the3-ketoacyl-CoA synthase, sub-sequence SEQ ID NO. 5 is preferably presentdownstream of sub-sequence SEQ ID NO. 1 or SEQ ID NO. 160, as the casemay be. The start of sub-sequence SEQ ID NO. 5 may be located, forexample, 100 to 120 amino acid residues, 108 to 116 amino acid residues,or exactly 112 or 113 amino acids, downstream of the start ofsub-sequence SEQ ID NO. 1 or SEQ ID NO. 160 (as the case may be) in such3-ketoacyl-CoA synthase enzymes.

In some embodiments in which SEQ ID NO. 6 or SEQ ID NO. 162 is presentin the 3-ketoacyl-CoA synthase, sub-sequence SEQ ID NO. 6 or SEQ ID NO.162, as the case may be, preferably is present downstream ofsub-sequence SEQ ID NO. 1 or SEQ ID NO. 160, as the case may be. Thestart of sub-sequence SEQ ID NO. 6 or SEQ ID NO. 162 may be located, forexample, from 140 to 160 amino acid residues, from 145 to 155 amino,especially exactly 147 or 148 amino acid residues, downstream of thestart of sub-sequence SEQ ID NO. 1 or SEQ ID NO. 160 (as the case maybe) in such 3-ketoacyl-CoA synthase enzymes.

In some embodiments in which both sub-sequences SEQ ID NOs. 5 and 6 arepresent, the 3-ketoacyl-CoA synthase includes sub-sequence SEQ ID NO.46. In SEQ ID NO. 46, sub-sequence SEQ ID NO. 5 appears as amino acidresidues 1-7 and sub-sequence SEQ ID NO. 6 appears as amino acidresidues 36-42.

In some embodiments in which both sub-sequences SEQ ID NOs. 5 and 162are present, the 3-ketoacyl-CoA synthase includes sub-sequence SEQ IDNO. 166. In SEQ ID NO. 166, sub-sequence SEQ ID NO. 5 appears as aminoacid residues 1-7 and sub-sequence SEQ ID NO. 162 appears as amino acidresidues 36-42.

In some embodiments in which each of sub-sequences SEQ ID NOs. 4, 5 and6 are present, the 3-ketoacyl-CoA synthase includes sub-sequence SEQ IDNO. 47. In SEQ ID NO. 47, sub-sequence SEQ ID NO. 4 appears as aminoacid residues 1-7: sub-sequence SEQ ID NO. 1 appears as amino acidresidues 28-36; sub-sequence SEQ ID NO. 5 appears as amino acid residues141-147 and sub-sequence SEQ ID NO. 6 appears as amino acid residues176-182.

In some embodiments in which each of sub-sequences SEQ ID NOs. 161, 5and 162 are present, the 3-ketoacyl-CoA synthase includes sub-sequenceSEQ ID NO. 167. In SEQ ID NO. 167, sub-sequence SEQ ID NO. 161 appearsas amino acid residues 1-7: sub-sequence SEQ ID NO. 160 appears as aminoacid residues 28-36; sub-sequence SEQ ID NO. 5 appears as amino acidresidues 141-147 and sub-sequence SEQ ID NO. 162 appears as amino acidresidues 176-182.

In any of the foregoing embodiments, the 3-ketoacyl-CoA synthase mayinclude sub-sequence SEQ ID NO. 48 or SEQ ID NO. 168. SEQ ID NO. 48 orSEQ ID NO. 168 preferably is upstream of sub-sequence SEQ ID NO. 1 orSEQ ID NO. 160, as the case may be and upstream of each of sub-sequencesSEQ ID NOs, 4 or 161, 5 and 6 or 162, when present.

In some embodiments, the 3-ketoacyl-CoA synthase includes SEQ ID. NO 49or a sequence that is at least 80%, at least 90% or at least 95%identical to SEQ ID NO. 49, provided that an amino acid residue thataligns to position 184 of SEQ ID NO. 49 is one of leucine, valine,isoleucine or methionine.

In some embodiments, the 3-ketoacyl-CoA synthase includes SEQ ID NO. 169or a sequence that is at least 80%, at least 90% or at least 95%identical to SEQ ID NO. 169, provided that an amino acid residue thataligns to position 184 of SEQ ID NO. 169 is one of cysteine, leucine,valine, isoleucine or methionine.

In some embodiments, the 3-ketoacyl-CoA synthase has SEQ ID. NO 110 oris at least 80%, at least 90% or at least 95% identical to SEQ ID NO.110, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue 184 of SEQ ID NO. 110 is one of leucine,valine, isoleucine or methionine.

In some embodiments, the 3-ketoacyl-CoA synthase has SEQ ID NO. 119 or asequence that is at least 80%, at least 90% or at least 95% identical toSEQ ID NO. 119, provided that an amino acid residue that aligns toposition 184 of SEQ ID NO. 119 is one of cysteine, leucine, valine,isoleucine or methionine.

In other embodiments, the 3-ketoacyl-CoA synthase is at least 50%, atleast 75%, at least 80%, at least 90% or at least 95% identical to anyof SEQ ID NOs. 8, 35, 86, 88 or 89, provided in each case that an aminoacid residue of the 3-ketoacyl-CoA synthase that aligns to amino acidresidue 184 of any of SEQ ID NOs. 8, 35, 86, 88 or 89 is leucine,valine, isoleucine or methionine.

In specific embodiments, the 3-ketoacyl-CoA synthase has any of SEQ IDNOs. 9-34, 36-39, 92-93, 121-157 or 172-204, or is at least 50%, atleast 75%, at least 80%, at least 90% or at least 95% identical to anyof SEQ ID NOs. 9-34, 36-39, 92-93, 121-157 or 172-204, provided in eachcase that an amino acid residue of the 3-ketoacyl-CoA that aligns toamino acid residue 184 of any of SEQ ID NOs. 9-34, 36-39, 92-93, 121-157or 172-204 is one of leucine, valine, isoleucine or methionine.

In certain embodiments, the 3-ketoacyl-CoA synthase includessub-sequence SEQ ID NO. 2, or a sequence at least 80% identical to SEQID NO. 2, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue 6 of SEQ ID NO. 2 is isoleucine ormethionine. A sub-sequence otherwise identical to SEQ ID NO. 2, but inwhich the amino acid residue aligning to amino acid residue 6 of SEQ IDNO. 2 is cysteine, appears in naturally-occuring 3-ketoacyl-CoAsynthases such as that of Pseudomonas stutzeri ATCC 17588 (SEQ ID NO.90, where such a sequence appears as amino acid residues 181-190 (withthe cysteine appearing as amino acid residue 186). This substitution ofthe cysteine has been found to shift fatty acid production (andproduction of fatty acid derivatives) toward C6, C8 and/or C10 fattyacids and derivatives, and away from longer-chain compounds.

As before, a 3-ketoacyl-CoA synthase that includes sub-sequence SEQ IDNO. 2 may include at least one sub-sequence selected from a) SEQ ID NO.4 or SEQ ID NO. 161, b) SEQ ID NO. 5 and c) SEQ ID NO. 6 or SEQ ID NO.162. The 3-ketoacyl-CoA synthase preferably includes each of thesesub-sequences. When sub-sequence SEQ ID NO. 4 or SEQ ID NO. 161 ispresent, sub-sequence SEQ ID NO. 4 or SEQ ID NO. 161 is present upstreamof subsequence SEQ ID NO. 2, and its start may be located, for example,25 to 35 amino acid residues, 28 to 32 amino acid residues, orespecially exactly 30 amino acid residues, upstream of the start ofsub-sequence SEQ ID NO. 2. When sub-sequence SEQ ID NO. 5 is present,sub-sequence SEQ ID NO. 5 preferably is present downstream ofsub-sequence SEQ ID NO. 2, and the start of sub-sequence SEQ ID NO. 5may be located, for example, 100 to 120 amino acid residues, from 110 to118, especially exactly 115 or 116 amino acid residues, downstream ofthe start of sub-sequence SEQ ID NO. 2. When sub-sequence SEQ ID NO. 6or SEQ ID NO. 162 is present, sub-sequence SEQ ID NO. 6 or SEQ ID NO.162 preferably is present downstream of sub-sequence SEQ ID NO. 2, andthe start of sub-sequence SEQ ID NO. 6 or SEQ ID NO. 162 may be located,for example, from 140 to 160 amino acid residues, from 145 to 155 aminoacid residue, especially exactly 148, 149 or 150 amino acid residues,downstream of the start of sub-sequence SEQ ID NO. 2 in such3-ketoacyl-CoA synthase enzymes.

In some embodiments in which sub-sequence SEQ ID NO. 2 is present, the3-ketoacyl-CoA synthase is at least 50%, at least 75%, at least 80%, atleast 90% or at least 95% identical to SEQ ID NO. 40, provided that anamino acid residue of the 3-ketoacyl-CoA synthase that aligns to aminoacid residue 186 of SEQ ID NO. 40 is isoleucine or methionine.

In certain embodiments, the 3-ketoacyl-CoA synthase includessub-sequence SEQ ID NO. 3, or a sequence at least 80% identical to SEQID NO. 3, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue 6 of SEQ ID NO. 3 is any one of isoleucine,methionine, leucine, threonine, cysteine, valine, glutamine,phenylalanine, aspartic acid, asparagine and tyrosine. A sub-sequenceotherwise identical to SEQ ID NO. 3, but in which the amino acid residuethat aligns to amino acid residue 6 of SEQ ID NO. 3 is alanine, appearsin naturally-occuring 3-ketoacyl-CoA synthases such as that ofAlishewanella agri BL06 (SEQ ID NO. 91, where such a sequence appears asamino acid residues 181-190 (with the alanine appearing as amino acidresidue 186). This substitution of the alanine has been found to sharplyreduce the production of C12 fatty acids and derivatives in favor of C6,C8 and/or C10 fatty acids and derivatives.

As before, a 3-ketoacyl-CoA synthase that includes sub-sequence SEQ IDNO. 3 may include at least one sub-sequence selected from a) SEQ ID NO.4 or SEQ ID NO. 161, b) SEQ ID NO. 5 and c) SEQ ID NO. 6 or SEQ ID NO.162. The 3-ketoacyl-CoA synthase preferably includes each of thesesub-sequences. When sub-sequence SEQ ID NO. 4 or SEQ ID NO 161 ispresent, sub-sequence SEQ ID NO. 4 or SEQ ID NO. 161 is present upstreamof subsequence SEQ ID NO. 3, and its start may be located, for example,25 to 35 amino acid residues, 28 to 32 amino acid residues, orespecially exactly 30 amino acid residues, upstream of the start ofsub-sequence SEQ ID NO. 3. When sub-sequence SEQ ID NO. 5 is present,sub-sequence SEQ ID NO. 5 preferably is present downstream ofsub-sequence SEQ ID NO. 3, and the start of sub-sequence SEQ ID NO. 5may be located, for example, 100 to 120 amino acid residues, from 110 to118, especially exactly 115 or 116 amino acid residues, downstream ofthe start of sub-sequence SEQ ID NO. 3. When sub-sequence SEQ ID NO. 6or SEQ ID NO. 162 is present, sub-sequence SEQ ID NO. 6 or SEQ ID NO.162 preferably is present downstream of sub-sequence SEQ ID NO. 3, andthe start of sub-sequence SEQ ID NO. 6 or SEQ ID NO. 162 may be located,for example, from 140 to 160 amino acid residues, from 145 to 155 aminoacid residue, especially exactly 148, 149 or 150 amino acid residues,downstream of the start of sub-sequence SEQ ID NO. 3 in such3-ketoacyl-CoA synthase enzymes.

In some embodiments in which sub-sequence SEQ ID NO. 3 is present, the3-ketoacyl-CoA synthase is at least 50%, at least 75%, at least 80%, atleast 90% or at least 95% identical to any of SEQ ID NO. 42, providedthat an amino acid residue of the 3-ketaoacyl-CoA synthase that alignsto amino acid residue 186 of SEQ ID NO. 42 is any one of isoleucine,methionine leucine, threonine, cysteine, valine, glutamine,phenylalanine, aspartic acid, asparagine and tyrosine.

In some embodiments in which sub-sequence SEQ ID NO. 3 is present, the3-ketoacyl-CoA synthase is at least 50%, at least 75%, at least 80%, atleast 90% or at least 95% identical to any of SEQ ID NO. 43, providedthat an amino acid residue of the 3-ketoacyl-CoA synthase that aligns toamino acid residue 186 of SEQ ID NO. 43 is isoleucine.

In some embodiments in which sub-sequence SEQ ID NO. 3 is present, the3-ketoacyl-CoA synthase has SEQ ID NO. 42 or SEQ ID NO. 43.

In some embodiments in which sub-sequence SEQ ID NO. 3 is present, the3-ketoacyl-CoA synthase has SEQ ID NO. 42, wherein:

a) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine;b) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine,and amino acid residue 241 is any one of methionine, phenylanaline,glutamic acid, leucine, tyrosine or aspartic acid;c) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine,and amino acid residue 239 is any one of glutamine, aspartic acid orasparagine;d) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine,and amino acid residue 246 is any of lysine or arginine;e) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine,amino acid residue 239 is any one of glutamine, aspartic acid orasparagine and amino acid residue 241 is any one of methionine,phenylanaline, glutamic acid, leucine, tyrosine or aspartic acid;f) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine,amino acid residue 239 is any one of glutamine, aspartic acid, tyrosineor asparagine and amino acid residue 246 is any one of lusine andarginine;g) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine,amino acid residue 241 is any one of methionine, phenylanaline, glutamicacid or aspartic acid and amino acid residue 246 is any one of lysineand arginine; orh) amino acid residue 186 is any one of isoleucine, threonine, cysteine,valine, glutamine, phenylalanine, aspartic acid, asparagine or tyrosine,amino acid residue 243 is glutamic acid and amino acid residue 246 isany one of histidine, lysine and arginine.

In some embodiments, the 3-ketoacyl-CoA synthase includes a sub-sequenceSEQ ID. NO. 170, or a sub-sequence at least 80% identical to SEQ ID NO.170, provided that (1) an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue 1 of SEQ ID NO. 170 is any one of alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, histidine,isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine,serine, threonine, tryptophan or tyrosine and (2) an amino acid residueof the 3-ketoacyl-CoA that aligns to amino acid residue 4 of SEQ ID NO.170 is valine or alanine. SEQ ID NO. 170 differs from SEQ ID NO. 171 inthat the glycine in amino acid position 1 of SEQ ID NO. 171 is replacedin SEQ ID NO. 170 with any one of alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, serine, threonine,tryptophan or tyrosine, and further in that the amino acid in position 4may be valine. SEQ ID NO. 171 represents a sub-sequence found in a rangeof naturally-occuring 3-ketoacyl-CoA synthases. SEQ ID NO. 171 appears,for example in the naturally-occuring 3-ketoacyl-CoA synthases ofAcinetobacter schindleri CIP 107287 (as amino acid residues 51-60 of SEQID NO. 8 with amino acid residue 55 being glutamic acid); Acinetobacterschindleri NIPH 900 (as amino acid residues 51-60 of SEQ ID NO. 86 withamino acid residue 55 being glutamic acid); Acinetobacter johnsii SH046(as amino acid residues 51-60 of SEQ ID NO. 35 with amino acid residue55 being glutamic acid); Acinetobacter lwoffii SH145 (as amino acidresidues 51-60 of SEQ ID NO. 88 with amino acid residue 5 being asparticacid); Acinetobacter sp NIPH 713 (as amino acid residues 51-60 of SEQ IDNO. 89 with amino acid residue 55 being glutamic acid).

In some embodiments, the 3-ketoacyl-CoA synthase includes a sub-sequenceSEQ ID. NO. 50, or a sub-sequence at least 80% identical to SEQ ID NO.50, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue 4 of SEQ ID NO. 50 is arginine. SEQ ID NO.50 differs from SEQ ID NO. 108 in that the lysine of SEQ ID NO. 108 isreplaced in SEQ ID NO. 50 with arginine. SEQ ID NO. 108 represents asub-sequence found in a range of naturally-occuring 3-ketoacyl-CoAsynthases. SEQ ID NO. 108 appears, for example in the naturally-occuring3-ketoacyl-CoA synthases of Acinetobacter schindleri CIP 107287 (asamino acid residues 275-281 of SEQ ID NO. 8 with amino acid residue 280being glutamine); Acinetobacter schindleri NIPH 900 (as amino acidresidues 275-281 of SEQ ID NO. 86 with amino acid residue 279 beingglutamine); Acinetobacter johnsii SH046 (as amino acid residues 275-281of SEQ ID NO. 35 with amino acid residue 280 being asparagine);Acinetobacter lwoffii SH145 (as amino acid residues 274-280 of SEQ IDNO. 88 with amino acid residue 279 being glutamine); Acinetobacter spNIPH 713 (as amino acid residues 274-280 of SEQ ID NO. 89 with aminoacid residue 279 being glutamine). This substitution of the lysine hasbeen found to increase fatty acid production rates (and production ratesof fatty acid derivatives).

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 50 insome embodiments includes at least one sub-sequence selected from SEQ IDNO. 51, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, or any combinationof any two or more thereof. The 3-ketoacyl-CoA synthase preferablyincludes each of sub-sequences SEQ ID NOs. 51, 4, 5 and 6. Whensub-sequence SEQ ID NO. 4 is present, it is preferably upstream fromsub-sequence SEQ ID NO. 50. The start of sub-sequence SEQ ID NO. 4 maybe, for example, from 120 to 130, 122 to 128, or 124 to 125 amino acidresidues upstream of the start of sub-sequence SEQ ID NO. 50. Whensub-sequence SEQ ID NO. 51 is present, it is preferably upstream fromsub-sequence SEQ ID NO. 50. The start of sub-sequence SEQ ID NO. 51 maybe, for example, 93 to 103, 95 to 100, or 97 to 98 amino acid residuesupstream of the start of sub-sequence SEQ ID NO. 50. When sub-sequenceSEQ ID NO. 5 is present, it is preferably downstream from sub-sequenceSEQ ID NO. 50. The start of sub-sequence SEQ ID NO. 5 may be, forexample, 10 to 20, 12-18, or exactly 15 amino acid residues downstreamof the start of sub-sequence SEQ ID NO. 50. When sub-sequence SEQ ID NO.6 is present, it is preferably downstream from sub-sequence SEQ ID NO.50. The start of sub-sequence SEQ ID NO. 5 may be, for example, 45 to56, 49 to 53, or exactly 51 amino acid residues downstream of the startof sub-sequence SEQ ID NO. 50.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 50 mayalso include any one or more of sub-sequence SEQ ID NO. 52, sub-sequenceSEQ ID NO. 53, sub-sequence SEQ ID NO. 46, sub-sequence SEQ ID NO. 48(provided amino acid residue 278 is arginine) or subsequence SEQ ID NO.110 (again provided amino acid residue 278 is arginine. The start ofsaid sub-sequence SEQ ID NO. 52 or 53 may be 120 to 130, 122 to 128 or124 to 126 amino acid residues upstream of the start of sub-sequence SEQID NO. 50. The start of sub-sequence SEQ ID NO. 52 may be 120 to 130,122 to 128 or 124-126 amino acid residues upstream of the start ofsub-sequence SEQ ID NO. 50. The start of sub-sequence SEQ ID NO. 46 maybe, for example, 10 to 20, 12-18, or exactly 15 amino acid residuesdownstream of the start of sub-sequence SEQ ID NO. 50. A 3-ketoacyl-CoAsynthase that includes sub-sequence SEQ ID NO. 50 may includesub-sequence SEQ ID NO. 54, provided that amino acid residue 129 of SEQID NO. 54 is arginine.

A 3-ketoacyl-CoA synthase sub-sequence that includes SEQ ID NO. 50 mayinclude SEQ ID NO. 55, or include a sequence at least 50%, at least 75%,at least 80%, at least 90% or at least 95% identical to SEQ ID NO. 55,provided that an amino acid residue of the 3-ketoacyl-CoA that aligns toamino acid residue 278 in SEQ ID NO. 55 is arginine. A 3-ketoacyl-CoAsynthase sub-sequence that includes SEQ ID NO. 50 may have SEQ ID NO.111, or be at least 50%, at least 75%, at least 80%, at least 90% or atleast 95% identical to SEQ ID NO. 111, provided that an amino acidresidue of the 3-ketoacyl-CoA that aligns to amino acid residue 278 inSEQ ID NO. 111 is arginine.

In some embodiments, the 3-ketoacyl-CoA synthase includes a sub-sequenceSEQ ID. NO. 56, or a sub-sequence at least 80% identical to SEQ ID NO.56, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue 6 of SEQ ID NO. 56 is alanine. As will beseen by comparing the sequences, SEQ ID NO. 56 differs from SEQ ID NO.109 in that the valine in amino acid residue position 6 of SEQ ID NO.109 is replaced in SEQ ID NO. 56 with alanine. SEQ ID NO. 109 representsa sub-sequence found in a range of naturally-occuring 3-ketoacyl-CoAsynthases. SEQ ID NO. 109 appears, for example in the naturally-occuring3-ketoacyl-CoA synthases of Acinetobacter schindleri CIP 107287 (asamino acid residues 291-300 of SEQ ID NO. 8 and Acinetobacter schindleriNIPH 900 (as amino acid residues 291-300 of SEQ ID NO. 86. Thissubstitution of the valine by alanine has been found to increase fattyacid production rates (and production rates of fatty acid derivatives).

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 56 insome embodiments includes at least one sub-sequence selected from SEQ IDNO. 51, SEQ ID NO. 4, and SEQ ID NO. 6, or any combination of any two ormore thereof. The 3-ketoacyl-CoA synthase preferably includes each ofsub-sequences SEQ ID NOs. 51, 4, and 6. The start of sub-sequence SEQ IDNO. 51 may be 109 to 120, 112 to 116 or 114 amino acid residues upstreamfrom the start of sub-sequence SEQ ID NO. 56. The start of sub-sequenceSEQ ID NO. 4 may be 135 to 148, 139 to 143 or 141 amino acid residuesupstream from the start of sub-sequence SEQ ID NO. 56. The start ofsub-sequence SEQ ID NO. 6 may be 30 to 40, 33 to 37 or 35 amino acidresidues downstream from the start of sub-sequence SEQ ID NO. 56.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 56 mayalso include any one or more of sub-sequence SEQ ID NO. 52, sub-sequenceSEQ ID NO. 53, or sub-sequence SEQ ID NO. 48. The start of sub-sequenceSEQ ID NO. 52 or 53 may be 135 to 148, 139 to 143 or 141 amino acidresidues upstream from the start of sub-sequence SEQ ID NO. 56. Thestart of sub-sequence SEQ ID NO. 48 may be 285 to 295, 288 to 292 or 290amino acid residues upstream from the start of sub-sequence SEQ ID NO.56. A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 56may include sub-sequence SEQ ID NO. 58.

A 3-ketoacyl-CoA synthase sub-sequence that includes SEQ ID NO. 56 mayhave SEQ ID NO. 59, or be at least 50%, at least 75%, at least 80%, atleast 90% or at least 95% identical to SEQ ID NO. 59, provided that anamino acid residue of the 3-ketoacyl-CoA that aligns to amino acidresidue 296 in SEQ ID NO. 59 is alanine. A 3-ketoacyl-CoA synthasesub-sequence that includes SEQ ID NO. 56 may have SEQ ID NO. 112, or beat least 50%, at least 75%, at least 80%, at least 90% or at least 95%identical to SEQ ID NO. 112, provided that an amino acid residue of the3-ketoacyl-CoA that aligns to amino acid residue 296 in SEQ ID NO. 112is alanine.

In some embodiments, the 3-ketoacyl-CoA synthase includes a sub-sequenceSEQ ID. NO. 60, or a sub-sequence at least 80% identical to SEQ ID NO.60, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue 2 of SEQ ID NO. 60 is leucine and an aminoacid residue of the 3-ketoacyl-CoA that aligns to amino acid residue 10of SEQ ID NO. 60 is glutamine. As will be seen by comparing thesequences, SEQ ID NO. 60 differs from SEQ ID NO. 116 in that themethionine in amino acid residue position 2 of SEQ ID NO. 116 isreplaced in SEQ ID NO. 60 with leucine. SEQ ID NO. 116 represents asub-sequence found in a range of naturally-occuring 3-ketoacyl-CoAsynthases. SEQ ID NO. 116 appears, for example in the naturally-occuring3-ketoacyl-CoA synthases of Acinetobacter schindleri CIP 107287 (asamino acid residues 177-190 of SEQ ID NO. 8 and Acinetobacter schindleriNIPH 900 (as amino acid residues 177-190 of SEQ ID NO. 86). Thissubstitution of the methionine by leucine has been found to increasefatty acid production rates (and production rates of fatty acidderivatives).

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 60 mayfurther include sub-sequence SEQ ID NO. 4. The start of sub-sequence SEQID NO. 4 may be 22 to 32, 25 to 29 or 27 amino acid residues upstream ofthe start of sub-sequence SEQ ID NO. 60. Such a 3-ketoacyl-CoA synthasemay have a sub-sequence SEQ ID NO. 61 or sub-sequence SEQ ID NO. 62.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 60 insome embodiments includes at least one sub-sequence selected from SEQ IDNO. 5 and SEQ ID NO. 6. The start of sub-sequence SEQ ID NO. 5 may be109 to 120, 11 to 115 or 112 to 113 amino acid residues downstream ofthe start of sub-sequence SEQ ID NO. 60. The start of sub-sequence SEQID NO. 6 may be 143 to 153, 146 to 151 or 148 to 149 amino acid residuesupstream of the start of sub-sequence SEQ ID NO. 60. The 3-ketoacyl-CoAsynthase preferably includes each of sub-sequences SEQ ID NOs. 4, 5 and6.

A 3-ketoacyl-CoA synthase that includes sub-sequences SEQ ID NO. 60, SEQID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 may include a sub-sequence SEQID NO. 63.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 60 mayalso include a sub-sequence SEQ ID NO. 48. The start of sub-sequence SEQID NO. 48 may be 173 to 183, 175 to 179 or 177 amino acid residuesupstream from the start of sub-sequence SEQ ID NO. 60. A 3-ketoacyl-CoAsynthase sub-sequence that includes SEQ ID NO. 60 may include SEQ ID NO.64, or include a sequences that is at least 50%, at least 75%, at least80%, at least 90% or at least 95% identical to SEQ ID NO. 64, providedthat amino acid residue in position 178 is leucine and the amino acidresidue in position 186 is glutamine. A 3-ketoacyl-CoA synthasesub-sequence that includes SEQ ID NO. 60 may have SEQ ID NO. 113, or beat least 50%, at least 75%, at least 80%, at least 90% or at least 95%identical to SEQ ID NO. 113, provided that amino acid residue inposition 178 is leucine and the amino acid residue in position 186 isglutamine.

In some embodiments, the 3-ketoacyl-CoA synthase includes a sub-sequenceSEQ ID. NO. 65, or a sub-sequence at least 80% identical to SEQ ID NO.65, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue in position 2 of SEQ ID NO. 65 is alanine.As will be seen by comparing the sequences, SEQ ID NO. 65 differs fromSEQ ID NO. 97 in that the valine in amino acid residue position 2 of SEQID NO. 97 is replaced in SEQ ID NO. 65 with alanine. SEQ ID NO. 97represents a sub-sequence found in a range of naturally-occuring3-ketoacyl-CoA synthases. SEQ ID NO. 97 appears, for example in thenaturally-occuring 3-ketoacyl-CoA synthases of Acinetobacter schindleriCIP 107287 (as amino acid residues 316-331 of SEQ ID NO. 8 with aminoacid residue 319 being leucine and amino acid residue 322 being asparticacid); Acinetobacter schindleri NIPH 900 (as amino acid residues 316-331of SEQ ID NO. 86 with amino acid residue 319 being leucine and aminoacid residue 322 being aspartic acid; Acinetobacter johnsii SH046 (asamino acid residues 316-331 of SEQ ID NO. 35 with amino acid residue 319being isoleucine and amino acid residue 322 being aspartic acid);Acinetobacter lwoffii SH145 (as amino acid residues 315-330 of SEQ IDNO. 88 with amino acid residue 318 being isoleucine and amino acidresidue 321 being asparagine); Acinetobacter sp NIPH 713 (as amino acidresidues 315-330 of SEQ ID NO. 89 with amino acid residue 318 beingmethionine and amino acid residue 321 being asparagine. Thissubstitution of the valine with alanine has been found to increase fattyacid production rates (and production rates of fatty acid derivatives).

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 65 mayfurther include sub-sequence SEQ ID NO. 5. The start of sub-sequence SEQID NO. 65 may be 20 to 31, 24 to 28 or 26 amino acid residues upstreamfrom the start of sub-sequence SEQ ID NO. 65. A 3-ketoacyl-CoA synthasethat includes sub-sequence SEQ ID NO. 65 and sub-sequence SEQ ID NO. 5may include sub-sequence SEQ ID NO. 66.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 65 mayfurther include sub-sequence SEQ ID NO. 51. The start of sub-sequenceSEQ ID NO. 51 may be 134-144, 136 to 140 or 138 to 139 amino acidresidues upstream from the start of sub-sequence SEQ ID NO. 65.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 65 mayinclude sub-sequence SEQ ID NO. 4. A 3-ketoacyl-CoA synthase thatincludes sub-sequence SEQ ID NO. 65 and sub-sequence SEQ ID NO. 4 mayinclude sub-sequence SEQ ID NO. 53 or sub-sequence SEQ ID NO. 67. Thestart of sub-sequence SEQ ID NO. 4, 53 or 67 may be 160 to 170, 163 to168 or 165 to 166 amino acid residues upstream from the start ofsub-sequence SEQ ID NO. 65.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 65 mayfurther include either sub-sequence SEQ ID NO. 48. The start ofsub-sequence SEQ ID NO. 48, may be 310 to 320, 313 to 316, or 314 to 315amino acid residues upstream from the start of sub-sequence SEQ ID NO.65. A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 65may include sub-sequence SEQ ID NO. 68 or a sub-sequence at least 50%,at least 75%, at least 80%, at least 90% or at least 95% identical toSEQ ID NO. 68, provided that an amino acid residue of the 3-ketoacyl-CoAthat aligns to amino acid residue 317 of SEQ ID NO. 68 is alanine. A3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 65 mayhave SEQ ID NO. 114 or be at least 50%, at least 75%, at least 80%, atleast 90% or at least 95% identical to SEQ ID NO. 114, provided that anamino acid residue of the 3-ketoacyl-CoA that aligns to amino acidresidue 317 of SEQ ID NO. 114 is alanine.

In some embodiments, the 3-ketoacyl-CoA synthase includes a sub-sequenceSEQ ID. NO. 69, or a sub-sequence at least 80% identical to SEQ ID NO.69, provided that an amino acid residue of the 3-ketoacyl-CoA thataligns to amino acid residue in position 6 of SEQ ID NO. 69 isisoleucine. As will be seen by comparing the sequences, SEQ ID NO. 69differs from SEQ ID NO. 107 in that the methionine in amino acid residueposition 6 of SEQ ID NO. 107 is replaced in SEQ ID NO. 69 withisoleucine. SEQ ID NO. 107 represents a sub-sequence found in a range ofnaturally-occuring 3-ketoacyl-CoA synthases. SEQ ID NO. 107 appears, forexample in the naturally-occuring 3-ketoacyl-CoA synthases ofAcinetobacter schindleri CIP 107287 (as amino acid residues 266-274 ofSEQ ID NO. 8 with amino acid residue 268 being valine and amino acidresidue 274 being lysine); Acinetobacter schindleri NIPH 900 (as aminoacid residues 266-274 of SEQ ID NO. 86 with amino acid residue 268 beingvaline and amino acid residue 274 being lysine); Acinetobacter johnsiiSH046 (as amino acid residues 266-274 of SEQ ID NO. 35 with amino acidresidue 268 being valine and amino acid residue 274 being alanine);Acinetobacter lwoffii SH145 (as amino acid residues 265-273 of SEQ IDNO. 88 with amino acid residue 267 being valine and amino acid residue273 being alanine); Acinetobacter sp NIPH 713 (as amino acid residues265-273 of SEQ ID NO. 89 with amino acid residue 267 being valine andamino acid residue 273 being alanine). This substitution of themethionine with isoleucine has been found to increase fatty acidproduction rates (and production rates of fatty acid derivatives).

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 69 mayfurther include sub-sequence SEQ ID NO. 51. The start of sub-sequenceSEQ ID NO. 51 may be 85 to 95, 87 to 91 or 88 to 89 amino acid residuesupstream from the start of sub-sequence SEQ ID NO. 69

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 69 mayfurther include any or all of sub-sequence SEQ ID NOs. 4, 5 and 6. A3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 69 andsub-sequence SEQ ID NO. 4 may include sub-sequence SEQ ID NO. 52 orsub-sequence SEQ ID NO. 53. A 3-ketoacyl-CoA synthase that includessub-sequence SEQ ID NO. 69, sub-sequence SEQ ID NO. 5 and sub-sequenceSEQ ID NO. 6 may include sub-sequence SEQ ID NO. 46. A 3-ketoacyl-CoAsynthase that includes sub-sequences SEQ ID NO. 69, 4, 5 and 6 mayinclude sub-sequence SEQ ID NO. 70.

A 3-ketoacyl-CoA synthase that includes sub-sequence SEQ ID NO. 69 mayinclude sub-sequence SEQ ID NO. 48. A 3-ketoacyl-CoA synthase thatincludes sub-sequence SEQ ID NO. 69 may include sub-sequence SEQ ID NO.71 or include a sub-sequence at least 50%, at least 75%, at least 80% atleast 90% or at least 95% identical to sub-sequence SEQ ID NO. 71,provided that an amino acid residue of the 3-ketoacyl-CoA that aligns toamino acid residue 271 of SEQ ID NO. 71 is isoleucine. Such a3-ketoacyl-CoA synthase may have SEQ ID NO. 115 or be at least 50%, atleast 75%, at least 80% at least 90% or at least 95% identical tosub-sequence SEQ ID NO. 115, provided that an amino acid residue of the3-ketoacyl-CoA that aligns to amino acid residue 271 of SEQ ID NO. 115is isoleucine.

Further Reaction Steps in the Non-Native Fatty Acid Synthesis Pathway

The reaction of acyl-CoA with malonyl-CoA produces a 3-ketoacyl-CoAcompound that must be reduced to the corresponding acyl compound beforeit can condense with another molecule of malonyl-CoA to extend thechain. The reduction takes place in three steps, the first being thereduction of the 3-ketoacyl group to the corresponding 3-hydroxyacylgroup. The second reaction is a dehydration to the corresponding3-enoylacyl compound, which is reduced in a third step to thecorresponding acyl-CoA. The first reaction step is enzymaticallycatalyzed by a keto-CoA reductase (KCR) enzyme (EC 1.1.1.35). The secondstep is enzymatically catalyzed by a 3-hydroxy-acyl-CoA dehydratase(3HDh) enzyme (EC 4.2.1.17). Some bifunctional enzymes catalyze both ofthe first and second step reactions (EC 1.1.1.35 and EC 4.2.1.55). Thethird reaction step is enzymatically catalyzed by an enoyl-CoA reductase(ECR) enzyme (EC 1.1.1.32).

Accordingly, the genetically modified cell preferably further comprisesat least one of (1) a heterologous KCR gene that encodes for a KCRenzyme; (2) a heterologous 3HDh gene that encodes for a 3HDh enzyme; (3)a heterologous gene that encodes for a bifunctional enzyme thatcatalyzes both of the first and second reaction steps (EC 1.1.1.35 and4.1.2.55) and (4) a heterologolous ECR gene that encodes for an ECRenzyme. Preferably, the genetically modified cell contains at least (1),(2) and (4) or at least (3) and (4). In each case, the gene preferablyis under the control of promoter and/or terminator sequences active inthe host cell.

The KCR enzyme may be, for example, one encoded by a P. aeruginosapafadB gene and/or having an amino acid sequence at least 80%, at least90%, at least 95%, at least 99% or 100% identical to SEQ. ID. NO. 103,one encoded by a P. aeruginosa fadG gene and/or having an amino acidsequence at least 80%, at least 90%, at least 95%, at least 99% or 100%identical to SEQ. ID. NO. 102, one encoded by a C. beijerinckii hbd geneand/or having an amino acid sequence at least 80%, at least 90%, atleast 95%, at least 99% or 100% identical to SEQ. ID. NO. 101, andothers as described in WO 2015/010103.

The 3HDh enzyme may be, for example, one encoded by a C. acetobutylicumcrt (short-chain-enoyl-CoA hydratase) gene and/or having an amino acidsequence at least 80%, at least 90%, at least 95%, at least 99% or 100%identical to SEQ. ID. NO. 99, one encoded by a P. putida ech (enoyl-CoAhydratase/aldolase) gene and/or having an amino acid sequence at least80%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ.ID. NO. 100, and others as described in WO 2015/010103.

Suitable bifunctional enzymes that catalyse both the first and secondreactions steps include, for example, one encoded by an E. coli fadBgene and/or having an amino acid sequence at least 80%, at least 90%, atleast 95%, at least 99% or 100% identical to SEQ. ID. NO. 98; oneencoded by an R. novegicus ech2 gene, and others as described in WO2015/010103.

Suitable ECR enzymes include, for example, one encoded by a T. denticolater gene and/or having an amino acid sequence at least 80%, at least90%, at least 95%, at least 99% or 100% identical to SEQ. ID. NO. 84.

The genetically modified cell of the invention in some embodimentsfurther includes at least one heterologous 3-ketobutyryl-CoA synthasegene, different from the modified 3-ketoacyl-CoA synthases describedabove, which encodes for a 3-ketobutyryl-CoA synthase. The heterologous3-ketobutyryl-CoA synthase gene may encode for a 3-ketobutyryl-CoAsynthase enzyme that is at least 80%, at least 90%, at least 95%, atleast 99% or 100% identical to any of those identified as SEQ ID NOs.1-120 of WO 2015/10103.

In some embodiments, the heterologous 3-ketobutyryl-CoA synthase gene isa Streptomyces Sp CL190 gene and/or a gene that encodes for an NphT7enzyme that is at least 80%, at least 90%, at least 95%, at least 99% or100% identical to SEQ ID NO. 83.

In some embodiments, the genetically modified cell of the inventionincludes at least one gene that encodes for a modified NphT7 enzyme asdescribed in WO 2015/10103. The modified NphT7 enzyme comprises an aminoacid sequence having at least 70% but less than 100% to SEQ ID NO. 83.The modified NphT7 enzyme may have, for example, one or more amino acidsubstitutions selected from the group consisting of H100L, I147T, F217V,Y144L, V157F, G309S, G288S, a PDRP to HFLQ substitution for amino acidresidues 86-89, I147F, I147M, 1147Q, I147S, I147C, 1147E, I147N, I147W,I147D, I147R, I147P, I147L, V196G, I147G, I147H, I147K, I147V, I147A,I147Y, F217G, F217A, F217L, F217I, F217M, F217T, F217P, F217S, F217E,F217L, F217V, F217W, S323A and S323V, and any combination of any two ormore thereof.

In some embodiments, the modified NphT7 enzyme comprises at least oneamino acid substitution selected from the group consisting of I147V,I147S, I147T, and at least one additional amino acid substitutionselected from H100L, F217V, S323A and S323V. In some embodiments, themodified NphT7 enzyme corresponds to SEQ ID NO. 82. In some embodiments,the modified NphT7 enzyme comprises an I147V, I147S or I147T amino acidsubstitution and an S323A amino acid substitution (corresponding to SEQID NO. 82 in which amino acid 100 is H, amino acid 147 is V, S or T,amino acid 217 is F and amino acid 323 is A). In some embodiment, themodified NphT7 enzyme comprises an H100L substitution, an I147V, I147Sor I147T amino acid substitution, an F217V substitution and an S323Aamino acid substitution (corresponding to SEQ ID NO. 82 in which aminoacid residue 100 is L, amino acid residue 147 is V, S or T, amino acidresidue 217 is V and amino acid residue 323 is A).

In certain embodiments, the genetically modified cell of the inventionincludes both of (1) a Streptomyces Sp CL190 nphT7 gene and/or a genethat encodes for an NphT7 enzyme that is at least 80%, at least 90%, atleast 95%, at least 99% or 100% identical to SEQ ID NO. 83 and (2) amodified NphT7 enzyme having one or more amino acid substitutionsselected from the group consisting of H100L, I147T, F217V, Y144L, V157F,G309S, G288S, a PDRP to HFLQ substitution for amino acid residues 86-89,I147F, I147M, I147Q, I147S, I147C, 1147E, I147N, I147W, I147D, I147R,I147P, I147L, V196G, I147G, I147H, I1147K, I147V, I147A, I147Y, F217G,F217A, F217L, F217I, F217M, F217T, F217P, F217S, F217E, F217L, F217V,F217W, S323A and S323V, and any combination of any two or more thereof.In preferred embodiments, the genetically modified cell includes a genethat encodes for an enzyme having SEQ ID NO. 83 and another gene thatencodes for an enzyme having SEQ ID. NO. 82. In especially preferredembodiments, the genetically modified cell includes a gene that encodesfor an enzyme having SEQ ID NO. 83 and another gene that encodes for anenzyme having SEQ ID. NO. 82 in which amino acid residue 100 is H or L,amino acid residue 147 is S, T or V, amino acid residue 271 is F or Vand amino acid residue 323 is A.

The genetically modified cell of the invention produces one or moreenzymes that terminate the acyl elongation cycle and produce a producthaving the desired chain length. Such a termination enzyme may or maynot be heterologous. The selection of termination enzyme may depend onwhether the desired product is a fatty acid or a derivative thereof suchas a fatty alcohol, a fatty aldehyde, a fatty alkene, a fatty amide, afatty ester or a fatty alkane.

The cell of the invention in some embodiments includes a heterologousthioesterase gene that encodes for a thioesterase such as an acyl-CoAesterase, in which case the product will be a fatty acid. Suitablethioesterases include those described in Table 11 of WO 2015/101013.

In some embodiments the cell of the invention includes a gene thatencodes for an ester synthase, in which case the product typically is afatty acid ester. Suitable ester synthases have amino acid sequences atleast 80%, at least 90%, at least 95%, at least 99% or at least 100%identical to any of the Marinobacter aquacolei Maq1 enzyme (SEQ ID NO.289 of WO 2015/10103), the Psychrobacter cryohaloentis Pcry1 enzyme (SEQID NO 290 of WO 2015/10103), the Rhodococcus jostii Rjos1 enzyme (SEQ IDNO 291 of WO 2015/10103), the, Alcanivorax borkumensis strain SK2 Abork1enzyme (SEQ ID NO 292 of WO 2015/10103) and the Hahella chejuensis hchegene (SEQ ID NO. 104). The ester synthase may have an amino acidsequence at least 80%, at least 90%, at least 95%, at least 99% or atleast 100% identical to the Hahella chejuensis Hche ester synthase (SEQID NO. 104).

The genetically modified cell of the invention may also include one ormore genes that encode for one or more of a fatty acyl-CoA reductase(alcohol or aldehyde forming), a fatty aldehyde reductase, an acyl-ACPreductase, an acyl-CoA:ACP acyltransferase, an acyl-CoA hydrolase, acarboxylic acid reductase, an aldehyde dehydrogenase and/or an acyl-ACPreductase.

The genetically modified cell of the invention also may include (A) oneor more genes that encode for a carboxyl transferase subunit a enzyme,(EC 6.3.1.2) such as an E. coli accA enzyme or an enzyme that is atleast 80%, at least 90%, at least 95% or at least 99% identical thereto;(B) one or more genes that encode for a biotin carboxyl carrier protein,(EC 6.4.1.2) such as an E. coli accB enzyme or an enzyme that is atleast 80%, at least 90%, at least 95% or at least 99% identical thereto;(C) one or more genes that encode for a biotin carboxylase subunitenzyme, (EC 6.3.4.14) such as an E. coli accC enzyme or an enzyme thatis at least 80%, at least 90%, at least 95% or at least 99% identicalthereto; (D) a carboxyl transferase subunit (3 (EC 6.4.1.2), such as anE. coli accD enzyme or an enzyme that is at least 80%, at least 90%, atleast 95% or at least 99% identical thereto, or a combination of any twoor more thereof. In some embodiments, all of (A)-(D) are present.

In some embodiments, the genetically modified cell of the inventionfurther comprises one or more additional genetic modifications thatfully or partially inhibit the production of one or more of thefollowing enzymes:

Methylglyoxal synthase (EC 4.2.3.3), for example that encoded by the E.coli mgsA gene.

Lactate dehydrogenase (EC 1.1.1.27), for example that encoded by the E.coli ldhA gene.

Phosphotransacetylase (EC 2.3.1.8), for example that encoded by the E.coli pta gene.

Acetate kinase (EC 2.7.2.1), for example that encoded by E. coli ackAgene.

Acyl-CoA synthase (EC 6.2.1.3), for example that encoded by the E. colifadD gene.

Pyruvate formate lyase (EC 2.3.1.54), for example that encoded by the E.coli pflB gene.

Pyruvate oxidase (EC 1.2.2.2), for example that encoded by the E. colipoxB gene.

Fused acetaldehyde-CoA dehydrogense (EC 1.2.1.10).

Trigger factor (EC 5.2.1.8), for example that encoded by the E. coli tiggene.

Restriction endonuclease (EC 3.1.21.3), for example that encoded by theE. coli hsdr514 gene.

The atoDAEB operon.

Acyl-CoA thioesterase (EC 3.1.2.-), for example that encoded by the E.coli tesB or yciA gene.

Acyl-coenzyme A dehydrogenase (EC 1.3.8.7), for example that encoded bythe E. coli fadE gene.

3-ketoacyl-CoA thiolase (EC 2.3.1.16), for example that encoded by theE. coli fadA gene.

L-ribulokinase (EC2.7.1.16), for example that encoded by the E. coliaraB gene.

L-ribulose-5-phosphate-4-epimerase (EC 5.1.3.4), for example thatencoded by the E. coli araD gene.

Beta-D-galactosidase (EC 3.2.1.23), for example that encoded by the E.coli lacZ gene.

Lambda phase lysogen.

Rhamnulose-1-phosphate aldolase (EC 4.1.2.19), for example that encodedby the E. coli rhaD gene.

Rhamnulokinase (EC 2.7.1.5), for example that encoded by the E. colirhaB gene.

F mating factor.

Rph-1 gene.

Other genetic modifications may be present in the cell, including any ofthose described in WO 2015/10103.

Any heterologous gene may be operatively linked to a promoter and/orterminator sequence that is functional in the host strain. The promotermay be an inducible promoter that functions only under certainconditions. For example, a low phosphate inducible promoter such as thepromoter of the wild-type E. coli phoE gene (PphoE) promoter) is auseful promoter for the 3-ketoacyl synthase gene. Such a promoter isactive in a low phosphate environment. Accordingly, a microorganism inwhich the 3-ketoacyl synthase gene of the invention is under the controlof an E. coli phoE promoter or another low phosphate inducible promotermay be cultivated in a fermentation medium containing no more than 25 mMphosphate, especially no more than 20 mM, no more than 2 mM, no morethan 1 mM, no more than 0.5 mM, or no more than 0.25 mM phosphate).

Any heterologous gene may be integrated into the genome of the hoststrain and/or present in one or more plasmids. If integrated into thegenome, the heterologous gene may be inserted at a targeted or randomlocation. Transformation methods such as electroporation and chemicalmethods (including calcium chloride and/or lithium acetate methods)known in the art are suitable. Examples of suitable transformationmethods are described, for example, in Molecular Cloning: A LaboratoryManual 4^(th) Ed. Spring Harbor Press 2012. In general, no specialtransformation methods are necessary to produce the genetically modifiedcells of the invention.

Deletions and/or disruptions of native genes can be performed bytransformation methods, by mutagenesis and/or by forced evolutionmethods. In mutagenesis methods, cells are exposed to ultravioletradiation or a mutagenic substance, under conditions sufficient toachieve a high kill rate (60-99.9%, preferably 90-99.9%) of the cells.Surviving cells are then plated and selected or screened for cellshaving the deleted or disrupted metabolic activity. Disruption ordeletion of the desired native gene(s) can be confirmed through PCR orSouthern analysis methods.

The genetically modified cells described herein are used to producecompounds having a straight-chain alkyl group. The cells are grown underconditions such that they produce such compounds, and the compounds arerecovered.

When the host cell is a plant cell, the plant can be grown and thecompound having the straight-chain alkyl group can be recovered from theplant or any portion thereof, such as roots, stems, leaves, flowers,seeds, seed pods and the like, in which the compound accumulates duringthe growth of the plant.

Single-cell and other microcells of the invention can be used in aculturing process to produce such compounds.

Culturing is performed generally by forming a culture medium thatincludes at least one carbon source that is capable of being metabolizedby the cell to produce the product compounds and nutrients as may berequired by the specific cell. The nutrients may include, for example,at least one nitrogen source such as yeast extract, peptone, tryptone,soy flour, corn steep liquor, or casein, at least one phosphorus source,one or more vitamins such as biotin, vitamin B12 and derivatives ofvitamin B12, thiamin, pantothenate, one or more trace metals and thelike. The fermentation medium may also contain additional materials suchas anti-foam agents, biocides, buffers and the like.

In some cases, such as the production of fatty acid esters, the culturemedium may also include a reagent that reacts with the straight-chaincompound to produce the desired product. In the specific case of fattyacid esters, for example the culture medium preferably contains analkanol such as methanol, ethanol or a C3-C8 alkanol. The alkanol reactsto produce the corresponding ester. A native or heterologous estersynthase, or other appropriate enzyme, may be expressed by the cell tocatalyze such a reaction.

Generally, the culture medium is inoculated with the cell of theinvention, and the inoculum is cultured in the medium so that the celldensity increases to a cell density suitable for production. The culturemedium is then maintained at conditions sufficient for the cells toproduce the desired product.

Suitable culture conditions will of course depend on requirement of theparticular host strain. The temperature of the culture medium may be,for example from 20° C. to 70° C., with a temperature of 25 to 40° C.being preferred for most cells.

The pH of the culture medium may be, for example, from 2.0 to 10.0, from3.0 to 9.0 or from 6.0 to pH 8.5.

It is contemplated that embodiments of the present invention may bepracticed using either batch, fed-batch or continuous processes and thatany known mode of bio-production would be suitable.

The culturing may be performed under aerobic, microaerobic, or anaerobicconditions, as required or can be tolerated by the particular cell.

Generally, no special culturing equipment is needed to perform thefermentation. The equipment may include, for example, a tank suitablefor holding the cell and the culture medium; a line for dischargingcontents from the culture tank to an extraction and/or separationvessel; and an extraction and/or separation vessel suitable for removalof the chemical product from cell culture waste.

The carbon source is one or more carbon-containing compounds that can bemetabolized by the cell of the invention as a source of carbon. Examplesof suitable carbon sources include sugars such as glucose, sucrose,fructose, lactose, C-5 sugars such as xylose and arabinose, glycerol andpolysaccharides such as starch and cellulose. Other suitable carbonsources include fermentable sugars as may be obtained from cellulosicand lignocellulosic biomass through processes of pretreatment andsaccharification, as described, for example, in U.S. Patent PublicationNo. 2007/0031918A1, hemicellulose, lignin, starch, oligosaccharidesand/or monosaccharides. Other suitable carbon sources includehigh-fructose corn syrup, cheese whey permeate, cornsteep liquor, sugarbeet molasses, and barley malt. Still other suitable carbon sourcesinclude carbon dioxide, carbon monoxide, methanol, methylamine andglucosamine.

The culturing process may be continued until a titer of the desiredproduct reaches at least 0.01, at least 0.05, at least 0.1, at least0.25, at least 0.5 or at least 1 g per liter of culture medium (g/L).The fermentation process may be continued until the titer reaches, forexample, up to 40, up to 45, up to 50, up to 80, up to 100, or up to 120g/L. The specific productivity may be, for example, from 0.01 and 0.60grams of the desired product per gram of cells on a dry weight basis perhour (g chemical product/g DCW-hr). The volumetric productivity achievedmay be at least 0.005 g of the desired product per liter per hour(g/L-hr), at least 0.01 g/L-hr, at least 0.1 g/L-hr or at least 0.5g/L-hr, and may be up to, for example, 10 g/L-hr, up to 5 g/L-hr or upto 1 g/L-hr. [0212] In some embodiments, specific productivity asmeasured over a 24-hour fermentation (culture) period may be greaterthan about 0.01, 0.05, 0.10, 0.20, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0,7.0, 8.0, 9.0, 10.0, 11.0 or 12.0 grams of chemical product per gram DCWof cells (based on the final DCW at the end of the 24-hour period).

The following examples are provided to illustrate the invention, but arenot intended to limit the scope thereof. All parts and percentages areby weight unless otherwise indicated.

EXAMPLES

The following host E. coli strains are used in the following examples asindicated.

Host Strain 1 is a mutant of the E. coli strain designated BW25113,available from the E. coli Genetic Strain Center (CGSC#7636; Dept. ofMolecular, Cellular, and Developmental Biology, Yale University, NewHaven, Conn.), having the following additional genetic modifications:

Designation Description ΔldhA::frt Deletion of native lactatedehydrogenase ΔpflB::frt Deletion of native pyruvate formate lyaseΔmgsA::frt Deletion of methylglyoxal synthase ΔpoxB::frt Deletion ofpyruvate oxidase Δpta-ack::frt Deletion of phosphotransacetylase andacetate kinase Δtig:frt Deletion of trigger factor protein ΔatoDAEB::frtDeletion to disrupt short-chain poly-(R)- 3-hydroxybutyrate synthesisΔfadD::frt Deletion of native acyl-CoA synthase ΔtesB::frt Deletion ofnative thioesterase ΔyciA:frt Deletion of native thioesterasefabI(ts)_zeo Insertion of E. coli fabI gene encoding a protein havingSEQ ID NO 85, except the serine at position 241 replaced byphenylalanine (S241F modification) and a zeomyin resistance marker atthe 3′ end and deletion of the wildtype fabI gene.

Host Strain 2 is a mutant of the BW 25113 E. coli strain with thefollowing additional genetic modifications:

Designation Description ΔldhA::frt Deletion of native lactatedehydrogenase Δpf1B::frt Deletion of native pyruvate formate lyaseΔmgsA::frt Deletion of methylglyoxal synthase ΔpoxB::frt Deletion ofpyruvate oxidase Δpta-ack::frt Deletion of phosphotransacetylase andacetate kinase Δtig:frt Deletion of trigger factor protein ΔatoDAEB::frtDeletion to disrupt short-chain poly-(R)- 3-hydroxybutyrate synthesisΔfadD::frt Deletion of native acyl-CoA synthase ΔtesB::frt Deletion ofnative thioesterase ΔyciA:Irt Deletion of native thioesterase ΔadheDeletion of native aldehyde-alcohol dehydrogenase fabI(ts) Insertion ofmodified E. coli fabI gene encoding an enzyme having SEQ ID NO. 85, withthe serine at position 241 replaced by phenylalanine (S241Fmodification), and deletion of the wildtype fabI gene P_(pstsIH)-nphT7-Insertion of a gene encoding for ter-TT-loxP Streptomyces Sp. CL190acetoacetyl-CoA synthase (NphT7, SEQ. ID. NO. 83) and a gene encodingfor Treponema denticola enoyl-CoA reductase (ter, SEQ. ID. NO. 84) undercontrol of E. coli pstsIH promoter and an E. coli terminator at locus ofnative adhE gene

Host Strain 3 is a mutant of the BW 25113 E. coli strain with thefollowing genetic modifications:

Designation Description ΔldhA::frt Deletion of native lactatedehydrogenase ΔpflB::frt Deletion of native pyruvate formate lyaseΔmgsA::frt Deletion of methylglyoxal synthase ΔpoxB::frt Deletion ofpyruvate oxidase Δpta-ack::frt Deletion of phosphotransacetylase andacetate kinase Δtig:frt Deletion of trigger factor protein ΔatoDAEB::frtDeletion to disrupt short-chain poly-(R)-3- hydroxybutyrate synthesisΔfadD::frt Deletion of native acyl-CoA synthase ΔtesB::frt Deletion ofnative thioesterase ΔyciA:frt Deletion of native thioesterase ΔadheDeletion of native aldehyde-alcohol dehydrogenase P_(pstsIH)-nphT7-Insertion of a gene encoding for Streptomyces Sp. CL190 ter-TT-loxPacetoacetyl-CoA synthase (NphT7, SEQ. ID. NO. 83) and a gene encodingfor Treponema denticola enoyl-CoA reductase (ter, SEQ. ID. NO. 84) undercontrol of E. coli pstsIH promoter and an E. coli terminator at locus ofnative adhE gene

Production of 3-Ketoacyl-CoA Synthase Genes and Mutants

3-ketoacyl-CoA synthase genes are synthesized based on publishedsequence information for various wild type 3-ketoacyl-CoA synthasegenes. Site-specific mutants of the synthesized 3-ketoacyl-CoA synthasegenes are generated by oligonucleotide-directed mutagenesis. The sourcesof the wild-type genes and the short-hand designations used herein foreach of them and the amino acid sequence of the native enzyme producedby the wild-type genes are as follows:

Enzyme Encoded Source Species Designation (wild-type strain)Acinetobacter schindleri CIP 107287 Asch SEQ ID NO. 8 Acinetobacterschindleri NIPH 900 Asch-2 SEQ ID NO. 86 Acinetobacter johnsonii SH046Ajoh-2 SEQ ID NO. 35 Acinetobacter lwoffii SH145 Alwo SEQ ID NO. 88Acinetobacter sp. NIPH 713 ANIP71 SEQ ID NO. 89 Pseudomonas stutzeriATCC 17588 Pstu SEQ ID NO. 90 Alishewanella agri BL06 Aagr SEQ ID NO. 91

In each case, the 3-ketoacyl-CoA synthase gene is fused to a DNAsequence encoding a protein fragment containing 6 histidine residues anda protease recognization site, and incorporated into a pET plasmidhaving a ColE1 origin of replication and a kanamycin resistance marker.

Mutations to the amino acid residues encoded by the wild-type genes aredesignated herein by the shorthand designation for the wild-type strain,followed in parenthesis by a 3-, 4- or 5 character code consisting of afirst letter designating the amino acid residue in the native enzyme, a1-, 2- or 3-digit number indicating the position of that amino acidresidue in the native enzyme, and a final letter designating the aminoacid residue in that position in the mutated enzyme. The single-letterdesignations are IUPAC amino acid abbreviations as reported, forexample, at Eur. J. Biochem. 138:9-37(1984). For example, thedesignation “Asch(T184I)” indicates that a threonine (T) at amino acidresidue position 184 in the wild type Acinetobacter schindleri CIP107287 enzyme has been replaced with an isoleucine (I).

Production of Multiply-Mutated 3-Ketoacyl-CoA Synthase Genes.

Multi-mutated genes are prepared from wild-type or mutated (parent)3-ketoacyl-CoA synthase genes using error-prone PCR as the mutagenicmethod. Error-prone PCR of the 3-ketoacyl-CoA synthase gene is carriedout using primers having SEQ ID NO. 94 and SEQ ID NO. 95 and EconoTaqDNA polymerase (Lucigen) with the thermocyling program: 94° C. 2 min,30×[94° C. 20s, 55° C. 20s, 72° C. 72s], 72° C. 10 min, 4° C. hold. Inaddition, error-prone PCR reactions contain 50, 100, 150 or 200 M MnCl₂.PCR fragments are purified with the DNA Clean and Concentrator kit (ZymoResearch), digested with DpnI at 37° C. for 1 h, and purified again. Theplasmid and insert are assembled using 2×HiFi Assembly Master Mix, atwo-fold molar excess of insert to plasmid and incubation at 50° C. for1 h.

The amino acid sequences of the gene produced by multiply-mutated genesare designated by a shorthand as described above, with the mutationslisted sequentially. For example, “Asch(T184I,S328V)” indicates that athreonine (T) at amino acid residue position 184 in the wild typeAcinetobacter schindleri CIP 107287 enzyme has been replaced with anisoleucine (I) and a serine at position 328 has been replaced withvaline.

Production of Mutant E. coli Strains

Mutant E. coli strains are prepared using standard electroporationmethods. In each case, the host strain is transformed with a “Type 1”plasmid and a “Type 2” plasmid as described below.

Type 1 plasmids are pACYC plasmids containing the p15a origin ofreplication and chloramphenicol resistance marker. The Type 1 plasmidsused in the following examples are:

Type 1A: this plasmid includes a mutated Streptomyces sp. nphT7 geneencoding for a 3-ketoacyl-CoA synthase having H100L, I147S, F217V andS323A mutations (the “LSVA” NphT7 mutant, SEQ ID NO. 82), an E. colibifunctional 3-hydroxyacyl-CoA dehydrogenase/dehydratase (fadB) gene anda T. denticola enoyl-CoA (ter) gene cassette, all under a native E. colipstsIH promoter and a native E. coli terminator. This plasmid alsocontains a Hahella chejuensis ester synthase gene fused to a DNAsequence encoding a protein fragment containing 6 histidine residues anda protease recognition site under an E. coli phoE promoter, and an ACC(acetyl-CoA carboxylase) cassette including fused E. coli accA and accDgenes with a E. coli tpiA promoter and a cassette including the E. coliaccB and E. coli accC genes under an E. coli rpiA promoter.

Type 1B: this plasmid includes a mutated Streptomyces sp. gene encodingfor an nphT7 enzyme having I147S andF217V modifications (the “SV” NphT7mutant, SEQ ID NO. 96) and a T. denticola enoyl-CoA (ter) gene cassette,under a native E. coli psts1H promoter and a native E. coli terminator.The plasmid further contains an E. coli bifunctional 3-hydroxyacyl-CoAdehydrogenase/dehydratase (fadB) gene under a native E. coli psts1Hpromoter; and a Hahella chejuensis ester synthase gene fused to a DNAsequence encoding a protein fragment containing 6 histidine residues anda protease recognition site under an E. coli phoE promoter.

Plasmid type 2A includes a ColE1 origin of replication, a kanamycinresistance marker and the 3-ketoacyl-CoA synthase gene to be evaluated(fused to a DNA sequence that encodes an N-terminal protein fragmentcontaining 6 histidine residues and a protease recognition site unlessindicated otherwise) under an E. coli promoter and an E. coliterminator. The E. coli promoter is either the promoter for the nativepstS gene (Ppsts1H promoter) or that for the native phoE gene (PphoEpromoter), the latter of which is a low phosphate inducible type. The3-ketoacyl-CoA synthase and the promoter for the 3-ketoacyl-CoA synthasegene are as indicated in the specific examples below.

Plasmid 2B includes a ColE1 origin of replication, a kanamycinresistance marker and the 3-ketoacyl-CoA synthase gene to be evaluated(in some cases without the His-tag) under the Ppsts1H (SEQ ID NO. 118)or PphoE promoter (SEQ ID NO. 117) and an E. coli terminator. The3-ketoacyl-CoA synthase and the promoter for its gene are as indicatedin the specific examples below.

Small-Scale Fermentation Method

A culture of synthetic medium containing salts, glucose, NH₄Cl, andsupplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20μg/ml chloramphenicol is inoculated with the strain to be tested andgrown overnight at 30° C. The OD₆₀₀ of a 1:10 dilution of this cultureis determined, and a volume of the original culture corresponding to 8OD units is centrifuged and the supernatant discarded. The pelletedcells are resuspended thoroughly in 4 mL fresh medium containing, 30 g/Lglucose, 0.158 mM phosphate (low phosphate medium), 1% (V/V) methanol(to produce fatty acid methyl esters) or ethanol (to produce fatty acidethyl esters), chloramphenicol, and kanamycin as above, and 1-mlaliquots dispensed into triplicate 16-mm glass tubes containing 64 μL ofheptadecane or methyl tetradecanoate. This represents a limitedphosphate medium that promotes the activity of a low phosphate induciblepromoter such as the E. coli PphoE promoter (SEQ ID NO. 117). The tubesare incubated at 30° C., 250 rpm for 4 hours. The incubation temperatureis then raised to 37° C. and incubation is continued for a further 20hours. The entire culture is extracted with methyl tert-butyl ether andthe extract analyzed for fatty acid esters by gas chromatography.

Small-Scale Fermentation Method Used for Examples 80-116

A culture of synthetic medium containing salts, glucose, NH₄Cl, andsupplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20μg/ml chloramphenicol is inoculated with the strain to be tested andgrown overnight at 32° C. The OD₆₀₀ of a 1:10 dilution of this cultureis determined in order to inoculate a Seed 2 flask to a final OD₆₀₀ of0.3. Seed 2 flasks contain 25-30 ml synthetic medium containing salts,glucose, NH₄Cl, and supplemented with vitamins, yeast extract, 35 g/mlkanamycin and 20 μg/ml chloramphenicol, 0-2% methanol. Seed 2 flasks areincubated at 32° C. for 6-7 hours.

The OD₆₀₀ of a 1:10 dilution of this culture is determined in order toinoculate a production flask to a final OD₆₀₀ of 0.01-0.025. Productionflasks contain 25 ml synthetic medium containing salts, glucose, NH₄Cl,and supplemented with vitamins, yeast extract, 35 μg/ml kanamycin and 20μg/ml chloramphenicol, 1.25-2.5 mM phosphate, 0-2% methanol, 3-6 g/lglucose, 15-40 g/l glycerol. This represents a limited phosphate mediumthat promotes the activity of a low phosphate inducible promoter such asthe E. coli PphoE promoter (SEQ ID NO. 117). Production flasks areincubated at 32° C. When phosphate is depleted the following additionsare made: 2 ml methyl myristate, 1 ml 12.5% tween 80, 1-1.25 ml 50%glycerol. Methanol (0-0.5 ml) is added at the beginning of theproduction assay or right after phosphate depletion. Additional methanolcan be added after phosphate depletion to compensate for methanolevaporation. Flasks are incubated at 35° C. for a further 24 hours.Samples at 24 hours are taken and extracted with 0.1% HCL in MTBE(Methyl tert butyl ether) and the extracts are analyzed for fatty acidesters by gas chromatography.

Small-Scale Fermentation Method Used for Examples 117-148

A shallow 96-well plate of synthetic medium containing salts, glucose,NH₄Cl, and supplemented with vitamins, yeast extract, 35 μg/ml kanamycinand 20 μg/ml chloramphenicol is inoculated with the strain to be testedand grown overnight at 30° C. The culture from the shallow 96-well plateis used to inoculate a deep well production plate with 2-3 μl per well.Each well of the production plate contains 400 μl synthetic mediumcontaining salts, glucose, NH₄Cl, and supplemented with vitamins, 35μg/ml kanamycin and 20 μg/ml chloramphenicol, 1.25 mM phosphate, 2%methanol, 3 g/l glucose, 30 g/l glycerol. This represents a limitedphosphate medium that promotes the activity of a low phosphate induciblepromoter such as the E. coli PphoE promoter (SEQ ID NO. 117). Productionplates are incubated at 32° C. When phosphate is depleted, 26 μl methylmyristate is added to each well. Plates are incubated at 35° C. for afurther 20 hours. Samples at 20 hours are taken and diluted withacetonitrile and analyzed for fatty acid esters by gas chromatography.

Examples 1-15 and Comparative Samples A-C-Asch(T184I) Variants

Mutant E. coli strain Examples 1 and 2 contain a modified Asch geneencoding a 3-ketoacyl-CoA synthase Asch(T184I) (SEQ ID NO. 9).Comparative Samples A and B contain the wild-type gene (encoding for SEQID NO. 8). Details of strain construction are as follows:

Plasmid 3-ketoacyl- Desig- Host Plasmid NphT7 2 CoA synthase nationStrain 1 Type mutant Type Promoter Mutant Ex. 1 1 1B SV 2A PpstsIHAsch(T184I) Comp. A* 1 1B SV 2A PpstsIH wt Asch Ex. 2 2 1A LSVA 2A PphoEAsch(T184I) Comp. B* 2 1A LSVA 2A PphoE wt Asch *Not an example of thisinvention.

Each of Examples 1 and 2 and Comparative Samples are cultured to producefatty acid methyl esters using the small-scale method described above.Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fattyacid esters produced are as indicated in the following table.Selectivities to C8 and C10 fatty acid esters are indicated under therespective “%” columns; these are calculated as the titer of the C8 orC10 fatty acid ester over the total fatty acid ester production. Amountsof higher- and lower-carbon number fatty acid esters are not shownseparately, but are included in the total FAME production valueindicated.

Total C6 C8 C10 3-ketoacyl- FAME pro- pro- pro- Desig- CoA productionduction duction duction nation synthase (g/L) g/L) g/L % g/L % Ex. 1Asch(T184I) 1.17 0.21 0.75 64.1 0.22 18.5 Comp. A* Wt** Asch 1.43 0.150.01 <1 1.27 88.5 Ex. 2 Asch(T184I) 1.21 0.35 0.68 56.2 0.14 11.4 Comp.B* Wt** Asch 2.13 0.27 0.07 3.1 1.75 82.1 *Not an example of thisinvention. **“Wt” in this and subsequent tables indicates the wildtypeenzyme.

The wildtype Asch enzyme produces about 82% C10 fatty acid methylesters, with minimal amounts of the C8 esters. By changing the threonineat position 184 to isoleucine, production is shifted from almostexclusively C10 fatty acid esters to mainly C8 fatty acid esters, withsome increased selectivity toward C6 fatty acid esters also being seen.The mutant strain Examples 1 and 2 are useful for producing a mixture offatty acid esters enriched in the C8 esters.

Mutant E. coli strain Examples 3-5 similarly contain a modified Aschgene encoding a mutated 3-ketoacyl-CoA synthase (Asch(T184L),Asch(T184M) or Asch(T184V)). Comparative Sample C again contains thewild-type gene. Details of strain construction are as follows:

Plasmid 3-ketoacyl- Desig- Host Plasmid NphT7 2 CoA synthase nationStrain 1 Type mutant Type Promoter Mutant Ex. 3 1 1B SV 2A PpstsIHAsch(T184L) Ex. 4 1 1B SV 2A PpstsIH Asch(T184M) Ex. 5 1 1B SV 2APpstsIH Asch(T184V) Comp. C* 1 1B SV 2A PpstsIH wt Asch *Not an exampleof this invention.

Each of Examples 3-5 and Comparative Sample C are cultivated to producefatty acid ethyl esters using the small-scale method described above.Total fatty acid ethyl ester (FAEE) and amounts of C6, C8 and C10 fattyacid esters produced are as indicated in the following table, as areselectivities to C8 and C10 fatty acid esters. Amounts of higher- andlower-carbon number fatty acid esters are not shown separately, but areincluded in the total FAEE production value indicated.

Total C10 3-ketoacyl- FAEE C6 CoA production production C8 productionproduction Designation synthase (g/L) g/L) g/L % g/L % Ex. 3 Asch(T184L)0.88 0.30 0.22 24.4 0.37 41.6 Ex. 4 Asch(T184M) 0.75 0.51 0.23 30.1 0.011.7 Ex. 5 Asch(T184V) 1.05 0.19 0.51 48.2 0.36 33.9 Comp. C wt Asch 1.220.13 0.01 0.7 1.09 88.7 *Not an example of this invention.

As seen above with Comparative Samples A and B, the wildtype Asch enzymeleads to mainly C10 fatty acid ester production. The T184L, T184M andT184V variations all shift production from C10 fatty acid esters tomainly C6 and C8 fatty acid esters. The T184M variation in Example 4reduces C10 fatty acid ester production to less than 2%. Selectivity toC8 fatty acid esters is almost 50% for the T184V variation.

Mutant E. coli strains Examples 6-15 similarly contain a mutated Aschgene encoding a mutated 3-ketoacyl-CoA synthase with multiple mutationsas indicated in the following table. Details of strain construction areas follows:

Desig- Host Plasmid NphT7 Plasmid 3-ketoacyl-CoA synthase nation Strain1 Type mutant 2 Type Promoter Mutant Ex. 6 1 1A LSVA 2A PpstsIHAsch(N152T, T184I) Ex. 7 1 1A LSVA 2A PpstsIH Asch(N152L, T184I) Ex. 8 11A LSVA 2A PpstsIH Asch(N152M, T184I) Ex. 9 1 1A LSVA 2A PpstsIHAsch(N152C, T184I) Ex. 10 1 1A LSVA 2A PpstsIH Asch(A69V, T184I, S328G)Ex. 11 1 1A LSVA 2A PpstsIH Asch(G111C, T184I, S328G) Ex. 12 1 1A LSVA2A PpstsIH Asch(D39V, T184I, S328G) Ex. 13 1 1A LSVA 2A PpstsIHAsch(D39V, G111C, T184I, S328G) Ex. 14 2 1A LSVA 2A PpstsIH Asch(T184I,V268A, V296A, S328G) Ex. 15 2 1A LSVA 2A PpstsIH Asch(T184I, V268A,K278R, S328G)

Each of Examples 6-15 are cultivated to produce fatty acid methyl estersusing the small-scale method described above. Total fatty acid methylester (FAME) and amounts of C6, C8 and C10 fatty acid esters producedare as indicated in the following table, as are selectivities to C8 andand C10 fatty acid esters. Amounts of higher- and lower-carbon numberfatty acid esters are not shown separately, but are included in thetotal FAME production value indicated.

Total FAME C6 C8 C10 3-ketoacyl-CoA production production productionproduction Designation synthase (g/L) g/L) g/L % g/L % Ex. 6 Asch(N152T,T184I) 0.91 0.11 0.53 58.2 0.27 29.3 Ex. 7 Asch(N152L, T184I) 0.41 0.120.23 57.3 0.05 12.2 Ex. 8 Asch(N152M, T184I) 1.01 0.12 0.57 56.3 0.3332.2 Ex. 9 Asch(N152C, T184I) 0.69 0.06 0.39 56.8 0.20 28.3 Ex. 10Asch(A69V, T184I, 0.67 0.04 0.53 79.7 0.05 7.5 S328G) Ex. 11 Asch(G111C,T184I, 0.58 0.05 0.45 78.1 0.03 5.7 S328G) Ex. 12 Asch(D39V, T184I, 0.610.04 0.48 79.6 0.04 7.0 S328G) Ex. 13 Asch(D39V, G111C, 0.60 0.08 0.4474.3 0.03 4.7 T184I, S328G) Ex. 14 Asch(T184I, V268A, 1.033 0.04 0.8479.8 0.12 13.2 V296A, S328G) Ex. 15 Asch(T184I, V268A, 1.375 0.01 0.9667.2 0.37 29.4 K278R, S328G)

As this data shows, the multiply-mutated enzymes that contain the T184mutations all shift production away from C10 fatty acid esters to mainlyC8 fatty acid esters. Mutated enzymes having the S328G variation areespecially selective toward C8 fatty acid esters in this evaluation,with selectivities approaching 80%.

Examples 16-18 and Comparative Sample D-Asch-2 Variants

Mutant E. coli strain Examples 16-18 contain a modified Asch-2 geneencoding a 3-ketoacyl-CoA synthase (Asch-2(T184M), Asch-2(T184V) andAsch-2(T184I), SEQ ID NOs. 22-24, respectively). Comparative Sample Dcontains the wild-type gene (encoding for SEQ ID NO. 86). Details ofstrain construction are as follows:

3-ketoacyl- Desig- Host Plasmid NphT7 Plasmid 2 CoA synthase nationStrain 1 Type mutant Type Promoter Mutant Comp. Host 1 1 A LSVA 2APpstsIH wt Asch- D* 2 Ex. 16 Host 1 1 A LSVA 2A PpstsIH Asch-2 (T184M)Ex. 17 Host 1 1 A LSVA 2A PpstsIH Asch-2 (T184V) Ex. 18 Host 1 1 A LSVA2A PpstsIH Asch-2 (T184I)

Each of Examples 16-18 and Comparative Sample D is cultivated to producefatty acid methyl esters using the small-scale method described above.Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fattyacid esters produced are as indicated in the following table, as areselectivities to C8 and C10 fatty acid esters. Amounts of higher- andlower-carbon number fatty acid esters are not shown separately, but areincluded in the total FAEE production value indicated.

Total FAME C6 C8 C10 3-ketoacyl- production production productionproduction Designation CoA synthase (g/L) g/L) g/L % g/L % Comp. D* wtAsch-2 2.75 0.03 0 0 2.66 96.7 Ex. 16 Asch-2 0.82 0.30 0.36 44.0 0.1113.8 (T184M) Ex. 17 Asch-2 1.66 0.07 0.57 34.1 0.97 58.7 (T184V) Ex. 18Asch-2 2.29 0.05 0.72 31.2 1.47 64.2 (T184I) *Not an example of thisinvention.

The wildtype Asch-2 gene produces C10 fatty acid esters almostexclusively. The T184 variations all shift production from C10 fattyacid esters towards C6 and C8 fatty acid esters. Selectivity to C8 fattyacid esters is increased from zero to about 30-45% with thesemodifications to the Asch-2 gene.

Examples 19-22 and Comparative Sample E—Alwo Variants

Mutant E. coli strain Examples 19-22 contain a modified Alwo geneencoding a mutated 3-ketoacyl-CoA synthase (Alwo(T184L), Alwo(T184M),Alwo(T184V) and Alwo(T184I), SEQ ID NOs. 26-29, respectively).Comparative Sample E contains the 5 wild-type gene (encoding for SEQ IDNO. 88). Details of strain construction are as follows:

Plasmid 3-ketoacyl- Desig- Host Plasmid NphT7 2 CoA synthase nationStrain 1 Type mutant Type Promoter Mutant Comp. E* 1 1B SV 2A PpstsIH wtAlwo Ex. 19 1 1B SV 2A PpstsIH Alwo(T184L) Ex. 20 1 1B SV 2A PpstsIHAlwo(T184M) Ex. 21 1 1B SV 2A PpstsIH Alwo(T184V) Ex. 22 1 1B SV 2APpstsIH Alwo(T184I)

Each of Examples 19-22 and Comparative Sample E are cultivated toproduce fatty acid ethyl esters using the small-scale method describedabove. Total fatty acid ethyl ester (FAEE) and amounts of C6, C8 and C10fatty acid esters produced are as indicated in the following table, asare selectivities to C8 and C10 fatty acid esters. Amounts of higher-and lower-carbon number fatty acid esters are not shown separately, butare included in the total FAEE production value indicated.

3-ketoacyl- Total FAEE C6 C8 C10 CoA production production productionproduction Designation synthase (g/L) g/L) g/L % g/L % Comp. E* wt Alwo0.95 0.05 0.00 0 0.89 93.7 Ex. 19 Alwo(T184L) 0.73 0.31 0.20 27.7 0.2229.6 Ex. 20 Alwo(T184M) 0.69 0.46 0.22 31.2 0.01 1.42 Ex. 21 Alwo(T184V)1.27 0.11 0.40 31.2 0.76 60.0 Ex. 22 Alwo(T184I) 0.85 0.19 0.53 61.80.13 15.5 *Not an example of this invention.

The effect of the T184 variations in the Alwo enzyme is similar to thoseseen in the Asch and Asch-2 mutations. Whereas the wildtype Alwo enzymeproduces over 90% C10 fatty acid esters and no C8 fatty acid esters, theT184L, M, V and I variations all shift production toward C6 and C8 fattyacid esters. The T184M and T184I variations are particularly effectivein this regard, with C10 fatty acid ester selectivity being reduced tobelow 20% in each of those cases and C8 fatty acid ester selectivityexceeding 60% in the T184I case.

Examples 23-26 and Comparative Sample F—Ajoh-2 Variants

Mutant E. coli strain Examples 23-26 contain a modified Ajoh-2 geneencoding a 3-ketoacyl-CoA synthase (Ajoh-2(T184L), Ajoh-2(T184M),Ajoh-2(T184V) and Ajoh-2(T184I), SEQ ID NOs. 36-39, respectively).Comparative Sample F contains the wild-type gene (encoding for SEQ IDNO. 35). Details of strain construction are as follows:

3-ketoacyl- Desig- Host Plasmid NphT7 Plasmid 2 CoA synthase nationStrain 1 Type mutant Type Promoter Mutant Comp. F* 1 1A LSVA 2A PpstsIHwt Ajoh-2 Ex. 23 1 1A LSVA 2A PpstsIH Ajoh-2 (T184L) Ex. 24 1 1A LSVA 2APpstsIH Ajoh-2 (T184M) Ex. 25 1 1A LSVA 2A PpstsIH Ajoh-2 (T184V) Ex. 261 1A LSVA 2A PpstsIH Ajoh-2 (T184I)

Each of Examples 23-26 and Comparative Sample F are cultivated toproduce fatty acid methyl esters using the small-scale method describedabove. Total fatty acid methyl ester (FAME) and amounts of C6, C8 andC10 fatty acid esters produced are as indicated in the following table,as are selectivities to C8 and C10 fatty acid esters. Amounts of higher-and lower-carbon number fatty acid esters are not shown separately, butare included in the total FAEE production value indicated.

Total 3-ketoacyl- FAME C6 C8 CoA production production production C10production Designation synthase (g/L) g/L) g/L % g/L % Comp. F* wtAjoh-2 2.65 0.09 0.00 0.0 2.50 96.0 Ex. 23 Ajoh-2 0.90 0.10 0.17 19.30.57 63.9 (T184L) Ex. 24 Ajoh-2 0.60 0.28 0.23 38.4 0.04 6.6 (T184M) Ex.25 Ajoh-2 2.38 0.15 0.59 24.9 1.58 66.3 (T184V) Ex. 26 Ajoh-2 0.97 0.120.39 39.9 0.42 43.4 (T184I) *Not an example of the invention.

The same general pattern is seen with the Ajoh-2 gene modifications. Thehigh selectivity of the wildtype gene to C10 fatty acid ester productionis shifted toward C6 and C8 fatty acid ester production. The T184M andT184I variations are particularly effective in this regard. The T184Vvariation results in a large increase 5 of overall productivity comparedto the T184L, M and I variations.

Examples 27-29 and Comparative Sample G—ANIP71 Variants

Mutant E. coli strain Examples 27-29 contain a modified ANIP71 geneencoding a 3-ketoacyl-CoA synthase (ANIP71(T184M), ANIP71(T184V) andANIP71(T184I), SEQ ID NOs. 32, 34 and 31, respectively). ComparativeSample G contains the wild-type gene (encoding for SEQ ID NO. 89).Details of strain construction are as follows:

3-ketoacyl- Desig- Host Plasmid NphT7 Plasmid 2 CoA synthase nationStrain 1 Type mutant Type Promoter Mutant Comp. G* 1 1A LSVA 2A PpstsIHwt ANIP71 Ex. 27 1 1A LSVA 2A PpstsIH ANIP71 (T184M) Ex. 28 1 1A LSVA 2APpstsIH ANIP71 (T184V) Ex. 29 1 1A LSVA 2A PpstsIH ANIP71 (T184I)

Each of Examples 27-29 and Comparative Sample G are cultivated toproduce fatty acid methyl esters using the small-scale method describedabove. Total fatty acid methyl ester (FAME) and amounts of C6, C8 andC10 fatty acid esters produced are as indicated in the following table,as are selectivities to C8 and C10 fatty acid esters. Amounts of higher-and lower-carbon number fatty acid esters are not shown separately, butare included in the total FAME production value indicated.

Total 3-ketoacyl- FAME C6 C8 C10 CoA production production productionproduction Designation synthase (g/L) g/L) g/L % g/L % Comp. G* wtANIP71 2.71 0.04 0 0 2.61 96.5 Ex. 27 ANIP71 0.49 0.27 0.15 30.1 0.036.1 (T184M) Ex. 28 ANIP71 2.46 0.10 0.58 23.4 1.72 70.0 (T184V) Ex. 29ANIP71 0.21 0.04 0.10 47.8 0.03 14.2 (T184I) *Not an example of theinvention.

As before, the T184 variations all shift production from C10 fatty acidesters towards C8 fatty acid esters, with the T184M and T184I variationsbeing particularly effective. The T184V variation results in a largeincrease of overall productivity compared with the T184M and T184Ivariations. The T184I variation exhibits the highest selectivity to C8fatty acid esters.

Examples 30-31 and Comparative Sample H—Pstu Variants

Mutant E. coli strain Examples 30-31 contain a modified Pstu geneencoding a 3-ketoacyl-CoA synthase (Pstu(C186M) and Pstu(C186I),respectively, SEQ ID NO. 40, with position 186 being M and I,respectively). Comparative Sample H contains the wild-type gene(encoding for SEQ ID NO. 90). Details of strain construction are asfollows:

Plasmid 3-ketoacyl- Desig- Host Plasmid NphT7 2 CoA synthase nationStrain 1 Type mutant Type Promoter Mutant Comp. H* 1 1A LSVA 2A PpstsIHwt Pstu Ex. 30 1 1A LSVA 2A PpstsIH Pstu (C186M) Ex. 31 1 1A LSVA 2APpstsIH Pstu (C186I)

Each of Examples 30-31 and Comparative Sample H are cultivated toproduce fatty acid methyl esters using the small-scale method describedabove. Total fatty acid methyl ester (FAME) and amounts of C6, C8 andC10 fatty acid esters produced are as indicated in the following table,as are selectivities to C8 and C10 fatty acid esters. Amounts of higher-and lower-carbon number fatty acid esters are not shown separately, butare included in the total FAME production value indicated.

Total 3-ketoacyl- FAME C6 C8 C10 CoA production production productionproduction Designation synthase (g/L) g/L) g/L % g/L % Comp. H* wt Pstu2.19 0.03 0 0 2.08 95.1 Ex. 30 Pstu 0.53 0.22 0.24 44.5 0.03 5.3 (C186M)Ex. 31 Pstu(C186I) 0.14 0.02 0.08 54.6 0 0 *Not an example of theinvention.

The wildtype Pstu enzyme produces C10 fatty acid esters almostexclusively. The C186M and I variations both shift production towards C6and C8 fatty acid esters, with selectivity toward C8 fatty acid estersbeing about 40-60%.

Examples 32-61 and Comparative Sample I—Aagr Variants

Mutant E. coli strains Examples 32-61 contain a modified Aagr geneencoding a 3-ketoacyl-CoA synthase, as indicated in the followingtables. Comparative Sample I contains the wild-type gene (encoding forSEQ ID NO. 91). Details of strain construction are as follows:

Host Plasmid NphT7 Plasmid 3-ketoacyl-CoA synthase Designation Strain 1Type mutant 2 Type Promoter Mutant Comp. I* 1 1B SV 1A PpstsIH wt AagrEx. 32 1 1B SV 1A PpstsIH Aagr(A186I) Ex. 33 1 1B SV 1A PpstsIHAagr(A186M) Ex. 34 1 1B SV 1A PpstsIH Aagr(A186L) Ex. 35 1 1B SV 1APpstsIH Aagr(A186T) Ex. 36 1 1B SV 1A PpstsIH Aagr(A186C) Ex. 37 1 1B SV1A PpstsIH Aagr(A186V) Ex. 38 1 1B SV 1A PpstsIH Aagr(A186Q) Ex. 39 1 1BSV 1A PpstsIH Aagr(A186F) Ex. 40 1 1B SV 1A PpstsIH Aagr(A186D) Ex. 41 11B SV 1A PpstsIH Aagr(A186N) Ex. 42 1 1B SV 1A PpstsIH Aagr(A186Y) Ex.43 1 1B SV 1A PpstsIH Aagr(A186I, I241D) Ex. 44 1 1B SV 1A PpstsIHAagr(A186I, I241E) Ex. 45 1 1B SV 1A PpstsIH Aagr(A186I, I241D, H246R)Ex. 46 1 1B SV 1A PpstsIH Aagr(A186I, I241E, H246R) Ex. 47 1 IB SV IAPpstsIH Aagr(A186I, C239N, H246R) Ex. 48 1 1B SV 1A PpstsIH Aagr(A186I,C239N, I241F) Ex. 49 1 1B SV 1A PpstsIH Aagr(A186I, C239N, I241Y) Ex. 501 1B SV 1A PpstsIH Aagr(A186I, C239D) Ex. 51 1 1B SV 1A PpstsIHAagr(A186I, I241L) Ex. 52 1 1B SV 1A PpstsIH Aagr(A186I, I241F) Ex. 53 11B SV 1A PpstsIH Aagr(A186I, I241Y) Ex. 54 1 1B SV 1A PpstsIHAagr(A186I, H246R) Ex. 55 1 1B SV 1A PpstsIH Aagr(A186I, H246K) Ex. 56 11B SV 1A PpstsIH Aagr(A186I, C239N) Ex. 57 1 1B SV 1A PpstsIHAagr(A186I, C239Q) Ex. 58 1 1B SV 1A PpstsIH Aagr(A186I, I241M) Ex. 59 11B SV 1A PpstsIH Aagr(A186I, I241D, H246K) Ex. 60 1 1B SV 1A PpstsIHAagr(A186I, I241Y, H246R) Ex. 61 1 1B SV 1A PpstsIH Aagr(A186I, I241E,H246K)

Each of Examples 32, 35-42, 44 and 50-61 and Comparative Sample I arecultivated to produce fatty acid ethyl esters in the small-scale methoddescribed above. Total fatty acid ethyl ester (FAEE) and amounts of C8and C10 fatty acid esters produced are as indicated below in thefollowing table, as are selectivities to C8, C10 and C12 fatty acidesters. Amounts of higher- and lower-carbon number fatty acid esters arenot shown separately, but are included in the total FAEE productionvalue indicated. “Small” under the “C12 Production” column means thatmost FAEE production not specifically accounted for in the table is theC6 ethyl ester, with less than 5% C12 fatty acid esters being produced.

Total FAEE C10 C12 Desig- 3-ketoacyl-CoA production C8 productionproduction production, nation synthase (g/L) g/L % g/L % % Comp. I* wtAagr 0.84 0.01 1.2 0.57 67.9 18.0 Ex. 32 Aagr(A186I) 1.32 0.40 22.7 0.6247.0 0.4 Ex. 35 Aagr(I186T) 1.16 0.02 1.9 0.81 69.8 11.0 Ex. 36Aagr(A186C) 0.96 0.01 1.5 0.64 67.0 13.4 Ex. 37 Aagr(A186V) 1.43 0.138.9 1.08 75.4 1.2 Ex. 38 Aagr(A186Q) 0.73 0.13 18.3 0.15 21.0 0.5 Ex. 39Aagr(A186F) 0.97 0.09 9.0 0 0.4 0.3 Ex. 40 Aagr(A186D) 0.55 0.01 2.10.40 72.5 7.8 Ex. 41 Aagr(A186N) 0.99 0.02 1.7 0.66 66.7 11.5 Ex. 42Aagr(A186Y) 0.62 0.07 10.8 0 0 0 Ex. 44 Aagr(A186I, I241E) 1.08 0.2220.7 0.60 55.5 Small Ex. 50 Aagr(A186I, C239D) 0.20 0.05 26.1 0.02 12.4Small Ex. 51 Aagr(A186I, I241L) 1.27 0.28 22.3 0.62 48.5 Small Ex. 52Aagr(A186I, I241F) 1.27 0.24 18.6 0.67 58.1 Small Ex. 53 Aagr(A186I,I241Y) 1.34 0.25 18.5 0.81 60.4 Small Ex. 54 Aagr(A186I, 1.22 0.36 29.50.60 49.0 Small H246R) Ex. 55 Aagr(A186I, H246K) 0.93 0.26 28.5 0.3538.1 Small Ex. 56 Aagr(A186I, C239N) 1.32 0.21 15.8 0.80 60.9 Small Ex.57 Aagr(A186I, C239Q) 0.11 0.03 28.5 0.02 14.3 Small Ex. 58 Aagr(A186I,I241M) 1.17 0.20 17.2 0.70 60.2 Small Ex. 59 Aagr(A186I, I241D, 0.360.13 36.7 0.10 26.1 Small H246K) Ex. 60 Aagr(A186I, I1241Y, 1.18 0.3327.6 0.68 57.2 Small H246R) Ex. 61 Aagr(A186I, I243E, 0.42 0.14 32.70.09 22.5 Small H246K) *Not an example of the invention.

The wildtype Aagr enzyme produces 18% C12 fatty acid esters in thisevaluation. The replacement of the wildtype Aagr reduces C12 fatty acidester production, in most cases in favor of higher selectivity toward C8and/or C10 fatty acid esters.

Each of Examples 32-34 and 43-49 are cultivated to produce fatty acidmethyl esters using the small-scale method described above. Total fattyacid methyl ester (FAME) and amounts of C8 and C10 fatty acid estersproduced are as indicated in the following table, as are selectivitiesto C8, C10 and C12 fatty acid esters. Amounts of higher- andlower-carbon number fatty acid esters are not shown separately, but areincluded in the total FAME production value indicated. “Small” under the“C12 Production” column means that most FAME production not accountedfor in the table is the C6 methyl ester, with less than 5% C12 fattyacid esters being produced.

Total FAME C10 C12 Desig- 3-ketoacyl-CoA production C8 productionproduction production, nation synthase (g/L) g/L % g/L % % Ex. 32Aagr(A186I) 1.46 0.20 14.0 1.06 72.8 0.5 Ex. 33 Aagr(A186M) 1.07 0.2623.9 0.1 9.33 Small Ex. 34 Aagr(A186L) 0.41 0.04 9.9 0.3 73.4 Small Ex.43 Aagr(A186I, I241D) 0.96 0.15 15.5 0.74 77.2 Small Ex. 44 Aagr(A186I,I241E) 0.99 0.13 12.8 0.81 81.6 Small Ex. 45 Aagr(A186I, I241D, 0.740.18 24.5 0.51 68.8 Small H246R) Ex. 46 Aagr(A186I, I241E, 0.42 0.1432.2 0.24 57.4 Small H246R) Ex. 47 Aagr(A186I, C239N, 0.09 0.04 48.80.04 41.7 Small H246R) Ex. 48 Aagr(A186I, C239N, 1.04 0.9 8.5 0.90 86.5Small I241F) Ex. 49 Aagr(A186I, C239N, 1.17 0.10 8.7 1.02 86.6 SmallI241Y)

As the data in the foregoing two table shows, the wild-type Aagr enzymeproduces a significant fraction (18%) of C12 fatty acid esters. TheA186I modification, by itself or accompanied by additionalmodifications, reduces C12 fatty acid ester production in favor of C8and/or C10 fatty acid esters, and in some cases also in favor of C6fatty acid esters, both in FAEE and FAME production.

Examples 62-71 and Comparative Samples J and K-Asch Variants

Mutant E. coli strains Examples 62-71 contain a modified Asch geneencoding a 3-ketoacyl-CoA synthase as indicated in the following tables.Comparative Samples J and K contain the wild-type gene (encoding for SEQID NO. 8). In Examples 62-64 and Comparative Sample J only, the mutantAsch gene is fused to a DNA sequence encoding a protein fragmentcontaining 6 histidine residues and a protease recognition site. Detailsof strain construction are as follows:

Plasmid Plasmid 3-ketoacyl- Desig- Host 1 NphT7 2 CoA synthase nationStrain Type mutant Type Promoter Mutant Comp. J. 2 1A LSVA 2A PpstsIH WtAsch Ex. 62 2 1A LSVA 2A PpstsIH Asch(K278R) Ex. 63 2 1A LSVA 2A PpstsIHAsch(V296A) Ex. 64 2 1A LSVA 2A PpstsIH Asch(K278R, V296A) Comp. K. 2 1ALSVA 2B PphoE Wt Asch Ex. 65 2 1A LSVA 2B PphoE Asch(V317A) Ex. 66 2 1ALSVA 2B PphoE Asch(K278R, V317A) Ex. 67 2 1A LSVA 2B PphoE Asch(V296A,V317A) Ex. 68 2 1A LSVA 2B PphoE Asch(M271I) Ex. 69 2 1A LSVA 2B PphoEAsch(M271I, V296A) Ex. 70 2 1A LSVA 2B PphoE Asch(M178L) Ex. 71 2 1ALSVA 2B PphoE Asch(M178L, V296A)

Each of 62-71 and Comparative Samples J and K are cultivated to producefatty acid methyl esters using the small-scale method described above.Total fatty acid methyl ester (FAME) and amounts of C6, C8 and C10 fattyacid esters produced are as indicated in the following table. Amounts ofhigher- and lower-carbon number fatty acid esters are not shownseparately, but are included in the total FAME production valueindicated.

Total C6 FAME pro- C8 C10 Desig- 3-ketoacyl-CoA production duction pro-pro- nation synthase (g/L) g/L) duction duction Comp. J Wt Asch 2.270.03 2.19 Ex. 62 Asch(K278R) 2.77 0.03 2.66 Ex. 63 Asch(V296A) 3.08 0.042.96 Ex. 64 Asch(K278R, V296A) 3.06 0.05 2.93 Comp. K. Wt Asch 4.18 0.230.03 3.87 Ex. 65 Asch(V317A) 4.66 0.29 0.04 4.27 Ex. 66 Asch(K278R,V317A) 4.33 0.29 0.04 3.95 Ex. 67 Asch(V296A, V317A) 4.89 0.26 0.03 4.55Ex. 68 Asch(M271I) 4.38 0.27 0.04 4.01 Ex. 69 Asch(M271I, V296A) 4.400.27 0.02 4.06 Ex. 70 Asch(M178L) 4.63 0.37 0.04 4.17 Ex. 71 Asch(M178L,V296A) 4.64 0.40 0.03 4.16

As this data shows, the K278R, the V296A, the V317A, the M271I and M178Lmutations all result in an increase in total productivity of the cell,compared to the wild-type Asch enzyme. Productivity is improved in theseexamples by as much as 36%. The generally higher productivity of Comp. Kand Examples 65-71 as compared to Comp. J and Examples 62-64 is believedto be attributable to the combination of having the 3-ketoacyl synthaseunder the control of a low phosphate inducible promoter together withthe selection of a low phosphate medium.

Examples 72-74-Asch(T184I) Variants

Mutant E. coli strains Examples 72-74 similarly contain a mutated Aschgene encoding a mutated 3-ketoacyl-CoA synthase with multiple mutationsas indicated in the following table. Details of strain construction areas follows:

Host Plasmid 1 NphT7 Plasmid 2 3-ketoacyl-CoA synthase DesignationStrain Type mutant Type Promoter Mutant Ex. 72 2 1A LSVA 2A PpstsIHAsch(T184I, V268A, V296A, S328G) Ex. 73 2 1A LSVA 2A PpstsIH Asch(T184I,V268A, V296A, V317A, S328G) Ex. 74 2 1A LSVA 2A PpstsIH Asch (V30A,T184I, V268A, V296A, V317A, S328G)

Each of examples 72-74 are cultivated to produce fatty acid methylesters using the small-scale method described above. Total fatty acidmethyl ester (FAME) and amounts of C6, C8 and C10 fatty acid estersproduced are as indicated in the following table, as are selectivitiesto C8 and and C10 fatty acid esters. Amounts of higher- and lower-carbonnumber fatty acid esters are not shown separately, but are included inthe total FAME production value indicated.

Total FAME C6 C8 C10 3-ketoacyl-CoA production production productionproduction Designation synthase (g/L) g/L) g/L % g/L % Ex. 72 Asch(T184I, 1.03 0.04 0.84 79.8 0.12 13.2 V268A, V296A, S328G) Ex. 73 Asch(T184I, 1.51 0.00 0.93 62 0.51 34 V268A, V296A, V317A, S328G) Ex. 74Asch (V30A, 1.47 0.05 1.17 80 0.19 11 T184I, V268A, V296A, V317A, S328G)

Examples 75-77

E. coli mutants are prepared by transforming E. coli strain BW25113 witha “Type 1” plasmid and a “Type 2” plasmid, using electroporation methodsdescribed before. Details of strain construction are as follows:

Example Plasmid NphT7 Plasmid 2 3-ketoacyl-CoA synthase No. 1 Typemutant Type Promoter Mutant Ex. 75 1B SV 2A PpstsIH Asch T184I Ex. 76 1ALSVA 2A PphoE Asch T184I Ex. 77 1A LSVA 2A PpstsIH Asch T184I

All of Examples 75-77 exhibit good selectivities toward C6-C10 fattyacid esters, when evaluated using the small-scale fermentation method.

Example 78

The Saccharomyces cerevisiae strain IMX581 (Mans, R., H. M. van Rossum,et al. (2015). CRISPR/Cas9: a molecular tool for simultaneousintroduction of multiple genetic modifications in Saccharomycescerevisiae. FEMS Yeast Res 15(2)) has Cas9 nuclease integrated in itschromosome such that it can be used as the host strain for manipulatingthe genome using CRISPR technology (US20140068797 A1). The guide RNA(gRNA) is expressed from either pMEL or pROS series of plasmids. Thegenes of the non-native fatty acid and fatty acid ester pathway areintegrated in the chromosome of IMX581 using this technology. The gRNAsequences are designed using Yeastriction online tool (Robert Mans,HEarme M. van Rossuni, Melanie Wijsman, Antoon Backx, Niels G A.Kuijpers, Marcel van den Broek, Pascale Daran-Lapujade, Jack T. Pronk,Anorionius J. A. van Maris, Jean-Marc G. Daran (2015) CRISPR/Cas9: amolecular Swiss army knife for simultaneous introduction of multiplegenetic modifications in Saccharomyces cerevisiae. FEMS Yeast Research16). The gRNA sequence is introduced into pMEL plasmid usingcomplementary primers that have 50 bp of homology and are PAGE-purified.The primers are dissolved in distilled water to a final concentration of100 μM, the primers are mixed in 1:1 molar ratio, and the mixture isheated to 95° C. for 5 min and annealed by cooling to room temperature.The primers are mixed with pMEL10 as template and the mixture isamplified using Q5 High Fidelity 2× Master Mix (New England BioLabs(Ipswich, Mass.). The PCR product is digested with DpnI for 30 minutesand the PCR product purified on an agarose gel. The protocol forsimultaneous integration and deletion is described in Mans et al(supra). Using the protocol, genes that encode for the proteins listedin the table below are integrated into loci in the S. cerevisiaechromosome as listed below. The terminators and promoters that are usedto express the genes are also listed in the table.

Enzyme Target Gene Encoded locus Promoter Terminator NphT7 SEQ ID NO. 83PDC1 gene Native PDC1 Native PDC1 NphT7 (LVSA) SEQ ID NO. 82 CIT3 geneNative TDH3 Native ADH1 variant Mutant 3-ketoacyl-CoA SEQ ID NO. 11 ADH1gene Native TEF1 Native ADH1 synthase 3-ketoacyl-CoA SEQ ID. NO. 98 GDH1gene Native PGK1 Native CYC1 reductase 3-hydroxyacyl-CoA reductase SEQID NO. 99 GAL1 gene Native GPD1 Native ADH1 Enoyl-CoA SEQ ID NO. 84GAL10 gene Native PGK1 Native CYC1 reductase Ester synthase SEQ ID NO.104 GPD1 gene Native GPD1 Native GPD1

The engineered yeast is grown in 250 mL shake flasks at 30° C. in 25 mLof synthetic defined medium supplemented with 10 g/L of glucose ascarbon source. The flasks are shaken at 200 rpm for 24 h. Fatty acid orfatty acid methyl ester acid is measured in the supernatant.

Example 79

The oleaginous yeast Yarrowia lipolytica strain LGAM S(7)1 (PapanikolaouS., and Aggelis G., Bioresour. Technol. 82(1):43-9 (2002)). CRISPR/Cas9:a molecular tool for simultaneous introduction of multiple geneticmodifications in Y. lipolytica. The host is engineered with Cas9nuclease integrated in its chromosome such that it can be used as thehost strain for manipulating the genome using CRISPR technology(US20140068797 A1). The guide RNA (gRNA) is expressed from either pMELor pROS series of plasmids. The genes of the non-native fatty acid andfatty acid ester pathway are integrated in the chromosome of Y.lipolytica using this technology. The gRNA sequences are designed usingYeastriction online tool (Robert Mans, Harmen M. van Rossum, MelanieWijsman, Antoon Backx, Niels G. A. Kuijpers, Marcel van den Broek,Pascale Daran-Lapujade, Jack T. Pronk, Antonius J. A. van Maris,Jean-Marc C. Daran (2015) CRISPR/Cas9: a molecular Swiss army knife forsimultaneous introduction of multiple genetic modifications inSaccharomyces cerevisiae. FEMS Yeast Research 16). The gRNA sequence isintroduced into pMEL plasmid using complementary primers that have 50 bpof homology and are PAGE-purified. The primers are dissolved indistilled water to a final concentration of 100 μM, the primers aremixed in 1:1 molar ratio, and the mixture is heated to 95° C. for 5 minand annealed by cooling to room temperature. The primers are mixed withpMEL10 as template and the mixture is amplified using Q5 High Fidelity2× Master Mix (New England BioLabs (Ipswich, Mass.). The PCR product isdigested with DpnI for 30 minutes and the PCR product purified on anagarose gel. The protocol for simultaneous integration and deletion isdescribed in Mans et al (supra). Using the protocol, genes that encodefor the proteins listed in the table below are integrated into the lociin the Y. lipolytica chromosome. Examples of the terminator andpromoters that are used to express the genes are also listed in thetable.

Enzyme Gene Encoded Target locus Promoter Terminator NphT7 SEQ ID NO. 83PDC1 gene Native PDC1 Native PDC1 NphT7 (LVSA) variant SEQ ID NO. 82XPR2 gene Native TDH3 Native ADH1 Mutant 3- SEQ ID NO. 11 ADH1 geneNative TEF1 Native ADH1 ketoacyl-CoA synthase 3-ketoacyl-CoA reductaseSEQ ID. NO. 98 GDH1 gene NativePGK1 Native CYC1 3-hydroxyacyl-CoA SEQ IDNO. 99 GAL1 gene Native GPD1 Native ADH1 reductase Enoyl-CoA reductaseSEQ ID NO. 84 GAL10 gene Native PGK1 Native CYC1 Ester synthase SEQ IDNO. 104 GPD1 gene Native GPD1 Native GPD1

The engineered yeast is grown in 250 mL shake flasks at 30° C. in 25 mLof synthetic defined media supplemented with 10 g/L of glucose as carbonsource. The 5 flasks are shaken at 200 rpm for 24 h. Fatty acid or fattyacid methyl ester acid is measured in the supernatant.

Examples 80-96-Asch(T184I) Variants

The mutant 3-ketoacyl-CoA synthase having SEQ ID NO. 20 is selected as apromising candidate for further improvement through additionalmutations. Inventive Control A is produced by introducing mutant3-ketoacyl-CoA synthase having SEQ ID NO. 20 into E. coli host strain 3.This inventive example serves as a basis for comparison for theadditional mutant Examples 80-96. Mutant E. coli strains Examples 80-96contain a mutated Asch gene encoding a mutated 3-ketoacyl-CoA synthasehaving the same mutations as SEQ ID NO. 20 together with one or moreadditional mutations. All mutations differing from the wild-type3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the followingtable. Details of strain construction are as follows:

Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant PromoterMutant Inventive 3 LSVA PphoE Asch (T184I, V268A, V296A, S328G), SEQControl A ID NO. 20 Ex. 80 3 LSVA PphoE Asch(T184I, V268A, V296A, K313E,S328G, A370T), SEQ ID NO. 121 Ex. 81 3 LSVA PphoE Asch(T18A, T184I,V268A, V296A, S328G), SEQ ID NO. 122 Ex. 82 3 LSVA PphoE Asch(T184I,V268A, V296A, S328G, S329G), SEQ ID NO. 123 Ex. 83 3 LSVA PphoEAsch(I127T, T184I, V268A, V296A, S328G), SEQ ID NO. 124 Ex. 84 3 LSVAPphoE Asch(T184I, V268A, K274E, V296A, S328G), SEQ ID NO. 125 Ex 85 3LSVA PphoE Asch(, T184I, N231I, V268A, V296A, ,S328G), SEQ ID NO. 204Ex. 86 3 LSVA PphoE Asch(A38V, M178T, T184I, V268A, V296A, A312D,S328G), SEQ ID NO. 126 Ex. 87 3 LSVA PphoE Asch(D116G, T184I, F190Y,L241P, V268A, V296A, S328G), SEQ ID NO. 127 Ex. 88 3 LSVA PphoEAsch(194T, T184I, V268A, V296A, S328G), SEQ ID NO. 128 Ex. 89 3 LSVAPphoE Asch(L22M, T184I, V268A, V296A, K313M, S328G), SEQ ID NO. 129 Ex.90 3 LSVA PphoE Asch(T184I, V268A, V296A, K313E, S328G), SEQ ID NO. 130Ex. 91 3 LSVA PphoE Asch(T184I, V268A, V296A, S328G, A370T), SEQ ID NO.131 Ex. 92 3 LSVA PphoE Asch(T18A, T184I, V268A, V296A, K313E, S328G,A370T), SEQ ID NO. 132 Ex. 93 3 LSVA PphoE Asch(T184I, V268A, V296A,K313E, S328G, S329G, A370T), SEQ ID NO. 133 Ex. 94 3 LSVA PphoEAsch(T184I, F236L, V268A, V296A, K313E, V317A, S328G), SEQ ID NO. 134Ex. 95 3 LSVA PphoE Asch(T184I, F236M, V268A, V296A, V317A, S328G), SEQID NO. 135 Ex. 96 3 LSVA PphoE Asch(T184I, I232V, V268A, V296A, S328G),SEQ ID NO. 136

Each of examples 80-96 are cultivated to produce fatty acid methylesters using the shake flask method described above. Amounts of C8 andC10 fatty acid esters are measured. The ability of each example toincrease production of C8 and/or C10 fatty esters is recorded asindicated. The ability of each example to increase specificity for C8and/or C10 is recorded as indicated.

Increase C8 Increase Increase C8 FAME total FAME FAME Specificityrelative to relative to relative to Inventive Inventive InventiveDesignation 3-ketoacyl-CoA synthase Control A Control A * Control A **Ex. 80 Asch(T184I, V268A, V296A, + ++ — K313E, S328G, A370T) Ex. 81Asch(T18A, T184I, V268A, + ++ + V296A, S328G) Ex. 82 Asch(T184I, V268A,V296A, ++ ++ — S328G, S329G) Ex. 83 Asch(I127T, T184I, V268A, +++ +++ OV296A, S328G) Ex. 84 Asch(T184I, V268A, K274E, ++ ++ O V296A, S328G) Ex.85 Asch(, T184I, N231I, V268A, +++ ++ O V296A, ,S328G) Ex. 86 Asch(A38V,M178T, T184I, + + O V268A, V296A, A312D, S328G) Ex. 87 Asch(D116G,T184I, F190Y, + + O L241P, V268A, V296A, S328G) Ex. 88 Asch(194T, T184I,V268A, ++ ++ + V296A, S328) Ex. 89 Asch(L22M, T184I, V268A, ++ ++ +++V296A, K313M, S328G) Ex. 90 Asch(T184I, V268A, V296A, + + + K313E ,S328G) Ex. 91 Asch(T184I, V268A, V296A, ++ ++ — S328G, A370T) Ex. 92Asch(T18A, T184I, V268A, + + — V296A, K313E, S328G, A370T) Ex. 93Asch(T184I, V268A, V296A, + + ++ K313E , S328G, S329G, A370T) Ex. 94Asch(T184I, F236L, V268A, + + +++ V296A, K313E, V317A, S328G) Ex. 95Asch(T184I, F236M, V268A, + V296A, V317A, S328G) Ex. 96 Asch(T184I,I232V, V268A, +++ +++ — V296A, S328G) *(+) = Increase over controlstrain **(+) = Increase over control strain; (—) = No change overcontrol strain; (O) = Decrease over control strain

Examples 97-99-Asch(T184I) Variants

A mutant 3-ketoacyl-CoA synthase having SEQ ID NO. 20, except that thevalines appearing at amino acids 30 and 317 each are replaced withalanine, is selected as a candidate for further improvement throughadditional mutations. Inventive Control B is produced by introducingthis mutant 3-ketoacyl-CoA synthase into E. coli host strain 2. Thisinventive example serves as a basis for comparison for the additionalmutants Examples 97-99. Mutant E. coli strains Examples 97-99 contain amutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase having thesame mutations as that of Inventive Control B together with one or moreadditional mutations. All mutations differing from the wild-type3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the followingtable. Details of strain construction are as follows:

Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant PromoterMutant Inventive 2 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, V317A,Control B S328G) Ex. 97 2 LSVA PpstsIH Asch(V30A, T184I, V268A, E282G,V296A, V317A, S328G), SEQ ID NO. 137 Ex. 98 2 LSVA PpstsIH Asch(V30A,T184I, V268A, V296A, V317A, D322G, S328G), SEQ ID NO. 138 Ex. 99 2 LSVAPpstsIH Asch(V30A, T184I, E210V, V268A, V296A, V317A, S328G), SEQ ID NO.139

Each of examples 97-99 are cultivated to produce fatty acid methylesters using the shake flask method described above. Amounts of C8 andC10 fatty acid esters are measured. The ability of each example toincrease production of C8 and/or C10 fatty esters is recorded asindicated. The ability of each example to increase specificity for C8and/or C10 is recorded as indicated.

Increase Increase Increase total C8 C8 FAME FAME FAME Specificityrelative relative relative to to to Inventive Design- InventiveInventive Control ation 3-ketoacyl-CoA synthase Control B Control B* B**Ex. 97 Asch(V30A, T184I, V268A, +++ ++ O E282G, V296A, V317A, S328G) Ex.98 Asch(V30A, T184I, V268A, +++ +++ — V296A, V317A, D322GS328G) Ex. 99Asch(V30A, T184I, E210V, + + — V268A, V296A, V317A, S328G) *(+) =Increase over control strain **(+) = Increase over control strain; (—) =No change over control strain; (O) = Decrease over control strain

Examples 100-116-Asch(T184I) Variants

A mutant 3-ketoacyl-CoA synthase corresponding to SEQ ID NO. 8 with sixspecific mutations as indicated in the following table, is selected as acandidate for further improvement through additional mutations.Inventive Control C is produced by introducing this mutant3-ketoacyl-CoA synthase into E. coli host strain 3. This inventiveexample serves as a basis for comparison for the additional mutantsExamples 100-116. Mutant E. coli strains Examples 100-116 contain amutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase having thesame mutations as that of Inventive Control C together with one or moreadditional mutations. All mutations differing from the wild-type3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the followingtable. Details of strain construction are as follows:

Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant PromoterMutant Inventive 3 LSVA PpstsIH Asch (V30A, T184I, V268A, V296A, V317A,Control C V328G) Ex. 100 3 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A,E315K, V317A, S328G, H368R), SEQ ID NO. 140 Ex. 101 3 LSVA PpstsIHAsch(V30A, A54V, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO.141 Ex. 102 3 LSVA PpstsIH Asch(V30A, T184I, V268A, V296A, 1302T, V317A,S328G, H368R), SEQ ID NO. 142 Ex. 103 3 LSVA PpstsIH Asch(V30A, A54V,T184I, V268A, V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 143 Ex. 1043 LSVA PpstsIH Asch(V30A, T184I, V268A, M271I, V296A, V317A, S328G,H368R), SEQ ID NO. 144 Ex. 105 3 LSVA PpstsIH Asch(V30A, T184I, V268A,V296A, V317A, S328G, A356G, H368R), SEQ ID NO. 145 Ex. 106 3 LSVAPpstsIH Asch(V30A, T184I, V268A, V296A, V317A, S328G, A3565, H368R), SEQID NO. 146 Ex. 107 3 LSVA PpstsIH Asch(V30A, A154G, T184I, V268A, V296A,V317A, S328G, H368R), SEQ ID NO. 147 Ex. 108 3 LSVA PpstsIH Asch(V30A,T184I, I232V, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 148 Ex. 1093 LSVA PpstsIH Asch(V30A, A54V, A154G, T184I, V268A, V296A, V317A,S328G, H368R), SEQ ID NO. 149 Ex. 110 3 LSVA PpstsIH Asch(V30A, A154G,T184I, V268A, M2711, V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 150Ex. 111 3 LSVA PpstsIH Asch(V30A, A54V, A154G, T184I, V268A, M271I,V296A, I302T, V317A, S328G, H368R), SEQ ID NO. 151 Ex. 112 3 LSVAPpstsIH Asch(V30A, A54V, T184I, V268A, M2711, V296A, I302T, V317A,S328G, H368R), SEQ ID NO. 152 Ex. 113 3 LSVA PpstsIH Asch(V30A, A54V,T184I, V268A, M2711, V296A, V317A, S328G, H368R), SEQ ID NO. 153 Ex. 1143 LSVA PpstsIH Asch(V30A, T184I, V268A, M271I, V296A, I302T, V317A,S328G, H368R), SEQ ID NO. 154 Ex. 115 3 LSVA PpstsIH Asch(V30A, A54V,A154G, T184I, V268A, M271I, V296A, V317A, S328G, H368R), SEQ ID NO. 155Ex. 116 3 LSVA PpstsIH Asch(V30A, A154G, T184I, V268A, M271I, V296A,V317A, S328G, H368R), SEQ ID NO. 156

Each of examples 100-116 are cultivated to produce fatty acid methylesters using the shake flask method described above. Amounts of C8 andC10 fatty acid esters are measured. The ability of each example toincrease production of C8 and/or C10 fatty esters is recorded asindicated.

Increase C8 Increase Increase C8 FAME total FAME FAME Specificityrelative to relative to relative to Inventive Inventive InventiveDesignation 3-ketoacyl-CoA synthase Control C Control C * Control C**Ex. 100 Asch(V30A, T184I, V268A, V296A, ++ + O E315K, V317A, S328G,H368R) Ex. 101 Asch(V30A, A54V, T184I, V268A, ++ ++ + V296A, V317A,S328G, H368R) Ex. 102 Asch(V30A, T184I, V268A, V296A, + + + I302T,V317A, S328G, H368R) Ex. 103 Asch(V30A, A54V, T184I, V268A, ++ ++ +V296A, I302TV317A, S328G, H368R) Ex. 104 Asch(V30A, T184I, V268A, M271I++ + + V296A, V317A, S328G, H368R) Ex. 105 Asch(V30A, T184I, V268A,V296A, +++ +++ + V317A, S328G, A356G, H368R) Ex. 106 Asch(V30A, T184I,V268A, V296A, + + + V317A, S328G, A356S, H368R) Ex. 107 Asch(V30A,A154G, T184I, V268A, ++ +++ + V296A, V317A, S328G, H368R) Ex. 108Asch(V30A, T184I, I232V, V268A, +++ ++ + V296A, V317A, S328G, H368R)Increase C8 Increase Increase C8 FAME total FAME FAME Specificityrelative to relative to relative to Designation 3-ketoacyl-CoA synthaseEx. 107 Ex. 107* Ex. 107** Ex. 109 Asch(V30A, A54V, A154G, T184I, + + OV268A, V296A, V317A, S328G, H368R) Ex. 110 Asch(V30A, A154G, T184I,V268A, + + — M271I, V296A, I302T, V317A, S3 28G, H368R) Ex. 111Asch(V30A, A54V, A154G, T184I, +++ +++ O V268A, M271I, V296A, I302T,V317A, S328G, H368R) Increase C8 Increase Increase C8 FAME total FAMEFAME Specificity relative to relative to relative to Designation3-ketoacyl-CoA synthase Ex. 104 Ex. 104* Ex. 104** Ex. 112 Asch(V30A,A54V, T184I, V268A, +++ +++ + M271I, V296A, I302T, V317A, S328G, H368R)Ex. 113 Asch(V30A, A54V, T184I, V268A, + + + M271I, V296A, V317A, S328G,H368R) Ex. 114 Asch(V30A, T184I, V268A, M271I, +++ +++ + V296A, I302T,V317A, S328G, H368R) Ex. 115 Asch(V30A, A54V, A154G, T184I, + + + V268A,M271I, V296A, V317A, S328G, H368R) Ex. 116 Asch(V30A, A154G, T184I,V268A, + ++ + M271IV296A, V317A, S328G, H368R) *(+) = Increase overcontrol strain **(+) = Increase over control strain; (—) = No changeover control strain; (O) = Decrease over control strain

Examples 117-133-Asch(G51) Variants

Mutant E. coli strains Examples 117-133 similarly contain a mutated Aschgene encoding a mutated 3-ketoacyl-CoA synthase with multiple mutationsas indicated in the following table. Details of strain construction areas follows:

Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant PromoterMutant Ex. 117 3 LSVA PphoE Asch(G51A, T184I, V268A, V296A, S328G), SEQID NO. 172 Ex. 118 3 LSVA PphoE Asch(G51C, T184I, V268A, V296A, S328G),SEQ ID NO. 173 Ex. 119 3 LSVA PphoE Asch(G51D, T184I, V268A, V296A,S328G), SEQ ID NO. 174 Ex. 120 3 LSVA PphoE Asch(G51H, T184I, V268A,V296A, S328G), SEQ ID NO. 175 Ex. 121 3 LSVA PphoE Asch(G51I, T184I,V268A, V296A, S328G), SEQ ID NO. 176 Ex. 122 3 LSVA PphoE Asch(G51K,T184I, V268A, V296A, S328G), SEQ ID NO. 177 Ex. 123 3 LSVA PphoEAsch(G51L, T184I, V268A, V296A, S328G), SEQ ID NO. 178 Ex. 124 3 LSVAPphoE Asch(G51M, T184I, V268A, V296A, S328G), SEQ ID NO. 179 Ex. 125 3LSVA PphoE Asch(G51N, T184I, V268A, V296A, S328G), SEQ ID NO. 180 Ex.126 3 LSVA PphoE Asch(G51P, T184I, V268A, V296A, S328G), SEQ ID NO. 181Ex. 127 3 LSVA PphoE Asch(G51Q, T184I, V268A, V296A, S328G), SEQ ID NO.182 Ex. 128 3 LSVA PphoE Asch(G51R, T184I, V268A, V296A, S328G), SEQ IDNO. 183 Ex. 129 3 LSVA PphoE Asch(G51S, T184I, V268A, V296A, S328G), SEQID NO. 184 Ex. 130 3 LSVA PphoE Asch(G51T, T184I, V268A, V296A, S328G),SEQ ID NO. 185 Ex. 131 3 LSVA PphoE Asch(G51V, T184I, V268A, V296A,S328G), SEQ ID NO. 186 Ex. 132 3 LSVA PphoE Asch(G51W, T184I, V268A,V296A, S328G), SEQ ID NO. 187 Ex. 133 3 LSVA PphoE Asch(G51Y, T184I,V268A, V296A, S328G), SEQ ID NO. 188

Each of examples 117-133 are cultivated to produce fatty acid methylesters using the shake flask method described above. Total fatty acidmethyl ester (FAME) and amounts of C8 and C10 fatty acid esters producedare as indicated in the following table, as are relative percentages ofC8 and and C10 fatty acids. Amounts of higher- and lower-carbon numberfatty acid esters are not shown separately, but are included in thetotal FAME production value indicated.

Total FAME production C8 FAME relative to g/L relative 3-ketoacyl-CoAInventive to Inventive Designation synthase Control A Control A Ex. 117Asch(G51A, + + T184I, V268A, V296A, S328G), SEQ ID NO. 172 Ex. 118Asch(G51C, + + T184I, V268A, V296A, S328G), SEQ ID NO. 173 Ex. 119Asch(G51D, +++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 174 Ex. 120Asch(G51H, ++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 175 Ex. 121Asch(G51I, + + T184I, V268A, V296A, S328G), SEQ ID NO. 176 Ex. 122Asch(G51K, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 177 Ex. 123Asch(G51L, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 178 Ex. 124Asch(G51M, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 179 Ex. 125Asch(G51N, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 180 Ex. 126Asch(G51P, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 181 Ex. 127Asch(G51Q, +++ +++ T184I, V268A, V296A, S328G), SEQ ID NO. 182 Ex. 128Asch(G51R, + + T184I, V268A, V296A, S328G), SEQ ID NO. 183 Ex. 129Asch(G51S, ++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 184 Ex. 130Asch(G51T, + ++ T184I, V268A, V296A, S328G), SEQ ID NO. 185 Ex. 131Asch(G51V, + ++ T184I, V268A, V296A, S328G), SEQ ID NO. 186 Ex. 133Asch(G51W, ++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 187 Ex. 133Asch(G51Y, +++ ++ T184I, V268A, V296A, S328G), SEQ ID NO. 188

Examples 134-148-Asch(G51) Variants

A mutant 3-ketoacyl-CoA synthase corresponding to SEQ ID NO. 8 withseven specific mutations as indicated in the following table, isselected as a candidate for further improvement through additionalmutations. Inventive Control D is produced by introducing this mutant3-ketoacyl-CoA synthase into E. coli host strain 3. This inventiveexample serves as a basis for comparison for the additional mutantsExamples 134-148. Mutant E. coli strains Examples 134-148 contain amutated Asch gene encoding a mutated 3-ketoacyl-CoA synthase having thesame mutations as that of Inventive Control D together with one or moreadditional mutations. All mutations differing from the wild-type3-ketoacyl-CoA synthase (SEQ ID NO. 8) are indicated in the followingtable. Details of strain construction are as follows:

Host NphT7 3-ketoacyl-CoA synthase Designation Strain mutant PromoterMutant Inventive 3 LSVA PpstsIH Asch (V30A, T184I, V268A, V296A, V317A,Control D S238G, H368R) Ex. 134 3 LSVA PpstsIH Asch(V30A, G51C, T184I,V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 189 Ex. 135 3 LSVAPpstsIH Asch(V30A, G51D, T184I, V268A, V296A, V317A, S328G, H368R), SEQID NO. 190 Ex. 136 3 LSVA PpstsIH Asch(V30A, G51E, T184I, V268A, V296A,V317A, S328G, H368R), SEQ ID NO. 191 Ex. 137 3 LSVA PpstsIH Asch(V30A,G51F, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 192 Ex. 1383 LSVA PpstsIH Asch(V30A, G51H, T184I, V268A, V296A, V317A, S328G,H368R), SEQ ID NO. 193 Ex. 139 3 LSVA PpstsIH Asch(V30A, G51I, T184I,V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 194 Ex. 140 3 LSVAPpstsIH Asch(V30A, G51K, T184I, V268A, V296A, V317A, S328G, H368R), SEQID NO. 195 Ex. 141 3 LSVA PpstsIH Asch(V30A, G51M, T184I, V268A, V296A,V317A, S328G, H368R), SEQ ID NO. 196 Ex. 142 3 LSVA PpstsIH Asch(V30A,G51N, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 197 Ex. 1433 LSVA PpstsIH Asch(V30A, G51P, T184I, V268A, V296A, V317A, S328G,H368R), SEQ ID NO. 198 Ex. 144 3 LSVA PpstsIH Asch(V30A, G51R, T184I,V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 199 Ex. 145 3 LSVAPpstsIH Asch(V30A, G515, T184I, V268A, V296A, V317A, S328G, H368R), SEQID NO. 200 Ex. 146 3 LSVA PpstsIH Asch(V30A, G51T, T184I, V268A, V296A,V317A, S328G, H368R), SEQ ID NO. 201 Ex. 147 3 LSVA PpstsIH Asch(V30A,G51W, T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 202 Ex. 1483 LSVA PpstsIH Asch(V30A, G51Y, T184I, V268A, V296A, V317A, S328G,H368R), SEQ ID NO. 203

Each of examples 134-148 are cultivated to produce fatty acid methylesters using the shake flask method described above. Total fatty acidmethyl ester (FAME) and amounts of C8 and C10 fatty acid esters producedare as indicated in the following table, as are relative percentages ofC8 and and C10 fatty acids. Amounts of higher- and lower-carbon numberfatty acid esters are not shown separately, but are included in thetotal FAME production value indicated.

Total FAME C8 FAME g/L production relative relative to to InventiveInventive Designation 3-ketoacyl-CoA synthase Control D Control D Ex.134 Asch(V30A, G51C, + +++ T184I, V268A, V296A, V317A, S328G, H368R),SEQ ID NO. 189 Ex. 135 Asch(V30A, G51D, +++ ++ T184I, V268A, V296A,V317A, S328G, H368R), SEQ ID NO. 190 Ex. 136 Asch(V30A, G51E, +++ ++T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 191 Ex. 137Asch(V30A, G51F, +++ ++ T184I, V268A, V296A, V317A, S328G, H368R), SEQID NO. 192 Ex. 138 Asch(V30A, G51H, + + T184I, V268A, V296A, V317A,S328G, H368R), SEQ ID NO. 193 Ex. 139 Asch(V30A, G51I, + +++ T184I,V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 194 Ex. 140 Asch(V30A,G51K, +++ +++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 195Ex. 141 Asch(V30A, G51M, ++ ++ T184I, V268A, V296A, V317A, S328G,H368R), SEQ ID NO. 196 Ex. 142 Asch(V30A, G51N, +++ ++ T184I, V268A,V296A, V317A, S328G, H368R), SEQ ID NO. 197 Ex. 143 Asch(V30A, G51P, + +T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 198 Ex. 144Asch(V30A, G51R, + + T184I, V268A, V296A, V317A, S328G, H368R), SEQ IDNO. 199 Ex. 145 Asch(V30A, G51S, + ++ T184I, V268A, V296A, V317A, S328G,H368R), SEQ ID NO. 200 Ex. 146 Asch(V30A, G51T, + +++ T184I, V268A,V296A, V317A, S328G, H368R), SEQ ID NO. 201 Ex. 147 Asch(V30A, G51W, ++++ T184I, V268A, V296A, V317A, S328G, H368R), SEQ ID NO. 202 Ex. 148Asch(V30A, G51Y, + + T184I, V268A, V296A, V317A, S328G, H368R), SEQ IDNO. 203

EMBODIMENTS

1. A 3-ketoacyl-CoA synthase having an amino acid sequence characterizedin including at least one of a) a sub-sequence at least 80% identical toSEQ ID NO. 1, provided that an amino acid residue that aligns to aminoacid residue 8 of SEQ ID NO. 1 is leucine, valine, isoleucine ormethionine and amino acid residue 2 is leucine or methionine; b) asub-sequence at least 80% identical to SEQ ID NO. 2, provided that anamino acid residue that aligns to amino acid residue 6 of SEQ ID NO. 2is isoleucine or methionine and c) a sub-sequence at least 80% identicalto SEQ ID NO. 3, provided that an amino acid residue that aligns withamino acid residue 6 of SEQ ID NO. 3 is isoleucine, methionine,threonine, cysteine, valine, glutamine, phenylalanine, aspartic acid,asparagine or tyrosine.2. A 3-ketoacyl-CoA synthase having an amino acid sequence characterizedin including at least one of a) SEQ ID NO. 1, b) SEQ ID NO. 2 and c) SEQID NO. 3.3. The 3-ketoacyl-CoA synthase of embodiment 2 wherein the amino acidsequence includes SEQ ID NO. 1.4. The 3-ketoacyl-CoA synthase of any of embodiments 1-3 wherein theamino acid sequence further includes at least one of a) SEQ ID NO. 4 orSEQ ID NO. 161, b) SEQ ID NO. 5 and c) SEQ ID NO. 6 or SEQ ID NO. 162.5. The 3-ketoacyl-CoA synthase of embodiment 4 wherein the amino acidsequence includes SEQ ID NO. 44.6. The 3-ketoacyl-CoA synthase of embodiment 5 wherein the amino acidsequence includes a sub-sequence at least 85% identical to SEQ ID NO.45, provided that an amino acid residue of the 3-ketoacyl-CoA synthasethat aligns to amino acid residue 35 of SEQ ID NO. 5 is leucine, valine,isoleucine or methionine.7. The 3-ketoacyl-CoA synthase of embodiment 5 wherein the amino acidsequence includes SEQ ID NO. 45.8. The 3-ketoacyl-CoA synthase of any of embodiments 4-7 wherein theamino acid sequence includes SEQ ID NO. 4 or SEQ ID NO. 161, SEQ ID NO.5 and SEQ ID NO. 6 or SEQ ID NO. 162.9. The 3-ketoacyl-CoA synthase of embodiment 7 wherein the amino acidsequence further includes SEQ ID NO. 46.10. The 3-ketoacyl-CoA synthase of embodiment 8 wherein the amino acidsequence includes SEQ ID NO. 47.11. The 3-ketoacyl-CoA synthase of any of embodiments 1-10 wherein theamino acid sequence includes SEQ ID NO. 48.12. The 3-ketoacyl-CoA synthase of embodiment 3 wherein the amino acidsequence is at least 80% identical to SEQ ID NO. 49.13. The 3-ketoacyl-CoA synthase of embodiment 3 wherein the amino acidsequence has SEQ ID NO. 49.14. The 3-ketoacyl-CoA synthase of any of embodiments 1-13 wherein theamino acid sequence is at least 50% identical to SEQ ID NO. 8.15. The 3-ketoacyl-CoA synthase of embodiment 14 wherein the amino acidsequence is at least 80% identical to SEQ ID NO. 8.16. A 3-ketoacyl-CoA synthase having an amino acid sequence is selectedfrom the group consisting of SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11,SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO.16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ IDNO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30,SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO.36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 92, SEQ IDNO. 93, any one of SEQ ID NOs 121-157 or any one of SEQ ID NOs. 172-204.17. The 3-ketoacyl-CoA synthase of embodiment 2 wherein the amino acidsequence includes SEQ ID NO. 2.18. The 3-ketoacyl-CoA synthase of embodiment 17 wherein the amino acidsequence further includes at least one of a) SEQ ID NO. 4 or SEQ ID NO.161, b) SEQ ID NO. 5 and c) SEQ ID NO. 6 or SEQ ID NO. 162.19. The 3-ketoacyl-CoA synthase of embodiment 18 wherein the amino acidsequence is at least 50% identical to SEQ ID NO. 40.20. The 3-ketoacyl-CoA synthase of embodiment 19 wherein the amino acidsequence is at least 80% identical to SEQ ID NO. 40.21. The 3-ketoacyl-CoA synthase of embodiment 18 which has SEQ. ID NO.40.22. The 3-ketoacyl-CoA synthase of embodiment 2 wherein the amino acidsequence includes SEQ ID NO. 3.23. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acidsequence further includes a) SEQ ID NO. 4 or SEQ ID NO. 161, b) SEQ IDNO. 5 and c) SEQ ID NO. 6 or SEQ ID NO. 162.24. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acidsequence includes SEQ ID NO. 41.25. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acidsequence is at least 50% identical to SEQ ID NO. 42 or 43.26. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acidsequence at least 80% identical to SEQ ID NO. 42 or 43.27. The 3-ketoacyl-CoA synthase of embodiment 22 wherein the amino acidsequence is SEQ. ID NO. 42 or 43.28. A 3-ketoacyl-CoA synthase having an amino acid sequencecharacterized in including SEQ ID. NO. 50.29. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acidsequence includes SEQ ID NO. 51.30. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acidsequence includes SEQ ID NO. 52.31. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acidsequence includes SEQ ID NO. 53.32. The 3-ketoacyl-CoA synthase of any of embodiments 23-31 wherein theamino acid sequence further includes SEQ ID NO. 46.33. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acidsequence includes SEQ ID NO. 54.34. The 3-ketoacyl-CoA synthase of any of embodiments 28-33 wherein theamino acid sequence further includes SEQ ID NO. 48.35. The 3-ketoacyl-CoA synthase of embodiment 28 wherein the amino acidsequence includes SEQ ID NO. 55.36. A 3-ketoacyl-CoA synthase having an amino acid sequencecharacterized in including SEQ ID. NO. 56.37. The 3-ketoacyl-CoA synthase of embodiment 36 wherein the amino acidsequence includes SEQ ID NO. 57.38. The 3-ketoacyl-CoA synthase of embodiment 36 or 37 wherein the aminoacid sequence further includes SEQ ID NO. 51.39. The 3-ketoacyl-CoA synthase of embodiment 36 or 37 wherein the aminoacid sequence further includes SEQ ID NO. 52.40. The 3-ketoacyl-CoA synthase of embodiment 36 or 37 wherein the aminoacid sequence further includes SEQ ID NO. 53.41. The 3-ketoacyl-CoA synthase of embodiment 36 wherein the amino acidsequence includes SEQ ID NO. 58.42. The 3-ketoacyl-CoA synthase of any of embodiments 36-41 wherein theamino acid sequence further includes SEQ ID NO. 48.43. The 3-ketoacyl-CoA synthase of embodiment 36 wherein the amino acidsequence includes SEQ ID NO. 59.44. A 3-ketoacyl-CoA synthase having an amino acid sequencecharacterized in including SEQ ID. NO. 60.45. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acidsequence includes SEQ ID. NO. 61.46. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acidsequence includes SEQ ID. NO. 62.47. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acidsequence includes SEQ ID. NO. 63.48. The 3-ketoacyl-CoA synthase of any of embodiments 44-46 wherein theamino acid includes SEQ ID. NO. 46.49. The 3-ketoacyl-CoA synthase of any of embodiments 44-48 wherein theamino acid sequence includes SEQ ID. NO. 48.50. The 3-ketoacyl-CoA synthase of embodiment 44 wherein the amino acidsequence includes SEQ ID. NO. 64.51. A 3-ketoacyl-CoA synthase having an acid sequence characterized inincluding SEQ ID. NO. 65.52. The 3-ketoacyl-CoA synthase of embodiment 51 wherein the amino acidsequence includes SEQ ID. NO. 51.53. The 3-ketoacyl-CoA synthase of any of embodiments 51 or 52 whereinthe amino acid sequence includes SEQ ID. NO. 66.54. The 3-ketoacyl-CoA synthase of any of embodiments 51-53 wherein theamino acid sequence includes SEQ ID. NO. 48.55. The 3-ketoacyl-CoA synthase of any of embodiments 51-54 wherein theamino acid sequence includes at least one of a) SEQ ID. NO. 4 or SEQ IDNO. 161 and b) SEQ ID NO. 5.56. The 3-ketoacyl-CoA synthase of any of embodiments 51-55 wherein theamino acid sequence includes SEQ ID. NO. 53.57. The 3-ketoacyl-CoA synthase of embodiment 51 wherein the amino acidsequence includes SEQ ID. NO. 67.58. The 3-ketoacyl-CoA synthase of embodiment 51 wherein the amino acidsequence includes SEQ ID. NO. 68.59. A 3-ketoacyl-CoA synthase comprising a heterologous nucleic acidsequence encoding a 3-ketoacyl-CoA synthase having an amino sequencecharacterized in including SEQ ID. NO. 69.60. The 3-ketoacyl-CoA synthase of embodiment 59 wherein the amino acidsequence further includes SEQ ID. NO. 51.61. The 3-ketoacyl-CoA synthase of embodiment 59 or 60 wherein the aminoacid sequence includes SEQ ID NO. 52.62. The 3-ketoacyl-CoA synthase of embodiment 59 or 69 wherein the aminoacid sequence includes SEQ ID NO. 53.63. The 3-ketoacyl-CoA synthase of any of embodiments 56-62 wherein theamino acid sequence includes at least one of a) SEQ ID NO. 5 and b) SEQID NO. 6 or SEQ ID NO. 162.64. The 3-ketoacyl-CoA synthase of any of embodiments 59-63 wherein theamino acid sequence includes SEQ ID NO. 46.65. The 3-ketoacyl-CoA synthase of embodiment 59 or 60 wherein the aminoacid sequence includes SEQ ID NO. 70.66. The 3-ketoacyl-CoA synthase of any of embodiments 59-65 wherein theamino acid sequence includes SEQ ID NO. 48.67. The 3-ketoacyl-CoA synthase of embodiment 59 wherein the amino acidsequence includes ID NO. 71.68. A ketoacyl-CoA synthase having SEQ ID NO. 110 or being at least 80%identical to SEQ ID NO. 110, comprising in each case at least one offeatures i)-xiii):

i) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 30 of SEQ ID NO. 110 is alanine;

ii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 39 of SEQ ID NO. 110 is valine;

iii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 69 of SEQ ID NO. 110 is valine;

iv) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 111 of SEQ ID NO. 110 is cysteine;

v) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 152 of SEQ ID NO. 110 is cysteine, leucine,methionine or threonine;

vi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 178 of SEQ ID NO. 110 is leucine;

vii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 184 of SEQ ID NO. 110 is isoleucine, leucine,methionine or valine;

viii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 268 of SEQ ID NO. 110 is alanine;

ix) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 271 of SEQ ID NO. 110 is isoleucine;

x) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 278 of SEQ ID NO. 110 is arginine;

xi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 296 of SEQ ID NO. 110 is alanine;

xii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 317 of SEQ ID NO. 110 is alanine;

xiii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 328 of SEQ ID NO. 110 is glycine.

69. A ketoacyl-CoA synthase having SEQ ID NO. 119 or being at least 80%identical to SEQ ID NO. 119, comprising in each case at least one offeatures i) to xliii):

i) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 18 of SEQ ID NO. 119 is alanine;

ii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 22 of SEQ ID NO. 119 is methionine;

iii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 30 of SEQ ID NO. 119 is alanine;

iv) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 38 of SEQ ID NO. 119 is valine;

v) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 39 of SEQ ID NO. 119 is valine;

vi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 51 of SEQ ID NO. 119 is alanine, cysteine, asparticacid, histidine, isoleucine, lysine, leucine, methionine, asparagine,proline, glutamine, arginine, serine, threonine, valine, tryptophan ortyrosine;

vii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 54 of SEQ ID NO. 119 is valine;

viii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 69 of SEQ ID NO. 119 is valine;

ix) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 83 of SEQ ID NO. 119 is asparagine;

x) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 94 of SEQ ID NO. 119 is threonine, leucine, glutamicacid, or alanine;

xi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 111 of SEQ ID NO. 119 is cysteine;

xii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 116 of SEQ ID NO. 119 is glycine;

xiii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 127 of SEQ ID NO. 119 is a threonine;

xiv) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 130 of SEQ ID NO. 119 is glycine;

xv) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 152 of SEQ ID NO. 119 is cysteine, leucine,methionine or threonine;

xvi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 154 of SEQ ID NO. 119 is glycine;

xvii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 178 of SEQ ID NO. 119 is leucine or threonine;

xviii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 190 of SEQ ID NO. 119 is phenylalanine ortyrosine;

xix) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 210 of SEQ ID NO. 119 is valine;

xx) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 223 of SEQ ID NO. 119 is histidine;

xxi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 231 of SEQ ID NO. 119 is isoleucine;

xxii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 232 of SEQ ID NO. 119 is valine;

xxiii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 236 of SEQ ID NO. 119 is leucine or methionine;

xxiv) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 241 of SEQ ID NO. 119 is proline;

xxv) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 268 of SEQ ID NO. 119 is alanine;

xxvi) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 271 of SEQ ID NO. 119 is isoleucine;

xxvii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 274 of SEQ ID NO. 119 is glutamic acid;

xxviii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 278 of SEQ ID NO. 119 is arginine, glutamicacid, aspartic acid, glutamine;

xxix) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 280 of SEQ ID NO. 119 is isoleucine;

xxx) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 282 of SEQ ID NO. 119 is glycine;

xxxi) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 296 of SEQ ID NO. 119 is alanine;

xxxii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 302 of SEQ ID NO. 119 is threonine;

xxxiii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 312 of SEQ ID NO. 119 is aspartic acid;

xxxiv) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 313 of SEQ ID NO. 119 is glutamic acid ormethionine;

xxxv) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 315 of SEQ ID NO. 119 is lysine;

xxxvi) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 317 of SEQ ID NO. 119 is alanine;

xxxvii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 322 of SEQ ID NO. 119 is glycine;

xxxviii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 328 of SEQ ID NO. 119 is glycine;

xxxix) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 329 of SEQ ID NO. 119 is glycine;

xl) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 344 of SEQ ID NO. 119 is glutamic acid, aspartic acidor asparagine; xli) an amino acid residue of the ketoacyl-CoA synthasethat aligns with amino acid residue 356 of SEQ ID NO. 119 is glycine orserine;

xlii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 368 of SEQ ID NO. 119 is arginine; and

xliii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 370 of SEQ ID NO. 119 is threonine.

70. The ketoacyl-CoA synthase of embodiment 69 wherein the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue184 of SEQ ID NO. 119 is isoleucine, leucine, methionine or valine.71. The ketoacyl-CoA synthase of embodiment 69 wherein the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue184 of SEQ ID NO. 119 is isoleucine.72. The ketoacyl-CoA synthase of any of embodiments 69-71 wherein:

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 268 of SEQ ID NO. 119 is alanine;

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 296 of SEQ ID NO. 119 is alanine; and

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 328 of SEQ ID NO. 119 is glycine.

73. The ketoacyl-CoA synthase of any of embodiments 69-72 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 317 of SEQ ID NO. 119 is alanine.74. The ketoacyl-CoA synthase of any of embodiments 70-73 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 30 of SEQ ID NO. 119 is alanine.75. The ketoacyl-CoA synthase of any of embodiments 70-74 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 368 of SEQ ID NO. 119 is arginine.76. The ketoacyl-CoA synthase of any of embodiments 69-75 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 51 of SEQ ID NO. 119 is alanine, cysteine, aspartic acid,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine.77. The ketoacyl-CoA synthase of any of embodiments 69-76 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 51 of SEQ ID NO. 119 is alanine.78. The 3-ketoacyl-CoA synthase having an amino acid sequence selectedfrom the group consisting of any of SEQ ID NOs. 121-157.79. A ketoacyl-CoA synthase having SEQ ID NO. 110 or being at least 80%identical to SEQ ID NO. 110, comprising in each case at least one offeatures i)-xiii):

i) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 30 of SEQ ID NO. 110 is alanine;

ii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 39 of SEQ ID NO. 110 is valine;

iii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 69 of SEQ ID NO. 110 is valine;

iv) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 111 of SEQ ID NO. 110 is cysteine;

v) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 152 of SEQ ID NO. 110 is cysteine, leucine,methionine or threonine;

vi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 178 of SEQ ID NO. 110 is leucine;

vii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 184 of SEQ ID NO. 110 is isoleucine, leucine,methionine or valine;

viii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 268 of SEQ ID NO. 110 is alanine;

ix) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 271 of SEQ ID NO. 110 is isoleucine;

x) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 278 of SEQ ID NO. 110 is arginine;

xi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 296 of SEQ ID NO. 110 is alanine;

xii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 317 of SEQ ID NO. 110 is alanine;

xiii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 328 of SEQ ID NO. 110 is glycine.

80. The ketoacyl-CoA synthase of embodiment 79 wherein the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue184 of SEQ ID NO. 120 is isoleucine, leucine, methionine or valine.81. The ketoacyl-CoA synthase of embodiment 79 wherein the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue184 of SEQ ID NO. 120 is isoleucine.82. The ketoacyl-CoA synthase of any of embodiments 79-81 wherein:

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 268 of SEQ ID NO. 120 is alanine;

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 296 of SEQ ID NO. 120 is alanine; and

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 328 of SEQ ID NO. 120 is glycine.

83. The ketoacyl-CoA synthase of any of embodiments 79-82 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 317 of SEQ ID NO. 120 is alanine.84. The ketoacyl-CoA synthase of any of embodiments 80-83 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 30 of SEQ ID NO. 120 is alanine.85. The ketoacyl-CoA synthase of any of embodiments 80-84 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 368 of SEQ ID NO. 120 is arginine.86. A ketoacyl-CoA synthase having SEQ ID NO. 110 or being at least 80%identical to SEQ ID NO. 110, comprising in each case at least one offeatures i)-xiii):

i) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 30 of SEQ ID NO. 110 is alanine;

ii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 39 of SEQ ID NO. 110 is valine;

iii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 69 of SEQ ID NO. 110 is valine;

iv) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 111 of SEQ ID NO. 110 is cysteine;

v) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 152 of SEQ ID NO. 110 is cysteine, leucine,methionine or threonine;

vi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 178 of SEQ ID NO. 110 is leucine;

vii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 184 of SEQ ID NO. 110 is isoleucine, leucine,methionine or valine;

viii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 268 of SEQ ID NO. 110 is alanine;

ix) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 271 of SEQ ID NO. 110 is isoleucine;

x) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 278 of SEQ ID NO. 110 is arginine;

xi) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 296 of SEQ ID NO. 110 is alanine;

xii) an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 317 of SEQ ID NO. 110 is alanine;

xiii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 328 of SEQ ID NO. 110 is glycine.

87. The ketoacyl-CoA synthase of embodiment 86 wherein the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue184 of SEQ ID NO. 110 is isoleucine, leucine, methionine or valine.88. The ketoacyl-CoA synthase of embodiment 86 wherein the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue184 of SEQ ID NO. 110 is isoleucine.89. The ketoacyl-CoA synthase of any of embodiments 86-88 wherein:

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 268 of SEQ ID NO. 110 is alanine;

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 296 of SEQ ID NO. 110 is alanine; and

the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 328 of SEQ ID NO. 110 is glycine.

90. The ketoacyl-CoA synthase of any of embodiments 86-89 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 317 of SEQ ID NO. 110 is alanine.91. The ketoacyl-CoA synthase of any of embodiments 87-90 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 30 of SEQ ID NO. 110 is alanine.92. The ketoacyl-CoA synthase of any of embodiments 87-91 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 368 of SEQ ID NO. 110 is arginine.93. A 3-ketoacyl-CoA synthase having an acid sequence characterized inincluding SEQ ID. NO. 170.94. A 3-ketoacyl-CoA synthase gene encoding for a 3-ketoacyl-CoAsynthase of any of embodiments 1-93.95. A genetically modified cell comprising a heterologous nucleic acidsequence encoding a 3-ketoacyl-CoA synthase of any of embodiments 1-93.96. A genetically modified cell comprising a 3-ketoacyl-CoA synthasegene of embodiment 94.97. The genetically modified cell of embodiment 95 or 96 wherein the3-ketoacyl-CoA synthase is malonyl-CoA-dependent.98. The genetically modified cell of any of embodiment 95-97 whichfurther comprises a heterologous nucleic acid sequence that encodes amalonyl-CoA-dependent 3-ketohexanoyl-CoA synthase.99. The genetically modified cell of embodiment 98 wherein theheterologous nucleic acid sequence that encodes a malonyl-CoA-dependent3-ketohexanoyl-CoA synthase includes SEQ ID. NO. 82.100. The genetically modified cell of embodiment 98 wherein theheterologous nucleic acid sequence that encodes a malonyl-CoA-dependent3-ketohexanoyl-CoA synthase includes SEQ ID. NO. 82 in which amino acidresidue 100 is leucine, amino acid residue 147 is serine, threonine orphenylalanine, amino acid residue 217 is valine and amino acid residue323 is valine.101. The genetically modified cell of any of embodiments 95-100 whichfurther comprises a heterologous nucleic acid sequence that encodes amalonyl-CoA-dependent 3-ketobutyryl-CoA synthase.102. The genetically modified cell of embodiment 101 wherein theheterologous nucleic acid sequence that encodes a malonyl-CoA-dependent3-ketobutyryl-CoA synthase includes SEQ ID. NO. 83.103. The genetically modified cell of any of embodiments 95-102 furthercomprising a heterologous nucleic acid sequence that encodes a3-ketoacyl-CoA reductase.104. The genetically modified cell of embodiment 103 wherein the3-ketoacyl-CoA reductase has or is at least 80% identical to any one ofSEQ. ID. NO. 103, SEQ. ID. NO. 102 or SEQ. ID. NO. 101.105. The genetically modified cell of any of embodiments 95-104 furthercomprising a heterologous nucleic acid sequence that encodes a3-hydroxyacyl-CoA dehydratase.106. The genetically modified cell of any of embodiments 95-105 furthercomprising a heterologous nucleic acid sequence that encodes for anenzyme that reduces a 3-ketoacyl-CoA to form a corresponding3-hydroxyacyl-CoA and dehydrates the 3-ketoacyl-CoA to form acorresponding 2-trans-enoyl-CoA.107. The genetically modified cell of embodiment 106 wherein theheterologous nucleic acid sequence that encodes for an enzyme thatreduces a 3-ketoacyl-CoA to form a corresponding 3-hydroxyacyl-CoA anddehydrates the 3-hydroxyacyl-CoA to form a corresponding2-trans-enoyl-CoA has or is at least 80% identical to SEQ. ID. NO. 98.108. The genetically modified cell of any of embodiments 95-107 furthercomprising a heterologous nucleic acid sequence that encodes anenoyl-CoA reductase.109. The genetically modified cell of any of embodiments 95-108 furthercomprising a heterologous nucleic acid sequence that encodes an estersynthase.110. The genetically modified cell of any of embodiments 95-109 furthercomprising a deletion of a native LDH gene.111. The genetically modified cell of any of embodiments 95-110 furthercomprising a deletion of a native pyruvate formate lyase gene.112. The genetically modified cell of any of embodiments 95-111 furthercomprising deletion of a native methylglyoxal synthase gene.113. The genetically modified cell of any of embodiments 95-112 furthercomprising a deletion of a native phosphotransacetylase gene.114. The genetically modified cell of any of embodiments 95-113 furthercomprising a deletion of a native thioesterase gene.115. The genetically modified cell of any of embodiments 95-114 furthercomprising a deletion of a native adhE gene.116. The genetically modified cell of any of embodiments 95-115 furthercomprising a deletion of a native atoDAEB operon.117. The genetically modified cell of any of embodiments 95-116 furthercomprising deletion of a native fadD gene.118. The genetically modified cell of any of embodiments 95-111 furthercomprising a deletion of a native phosphotransacetylase gene.119. The genetically modified cell of any of embodiments 95-118 furthercomprising a heterologous nucleic acid sequence that encodes for any ofa fatty acyl-CoA reductase, a fatty aldehyde reductase, an acyl-ACPreductase, an acyl-CoA:ACP acyltransferase, a thioesterase, an acyl-CoAhydrolase, a carboxylic acid reductase, a CoA hydrolase, an aldehydedehydrogenase, a carboxylic acid reductase and an acyl-ACP reductase.120. The genetically modified cell of any of embodiments 95-119 which isa bacteria.121. The genetically modified cell of embodiment 120 wherein thebacteria is E. coli.122. A process for making one or more compounds having a straight-chainalkyl group, comprising culturing the genetically modified cell of anyof embodiments 95-121 in a fermentation medium and recovering thecompound(s) having a straight-chain alkyl group from the fermentationmedium.123. The process of embodiment 122 wherein at least 40% by weight of thecompound(s) having a straight-chain alkyl group have 6-10 carbon atomsin the straight-chain alkyl group.124. The process of embodiment 122 wherein at least 60% by weight of thecompound(s) having a straight-chain alkyl group have 6-10 carbon atomsin the straight-chain alkyl group.125. The process of any of embodiments 122-124 wherein the compound(s)having a straight-chain alkyl group are fatty alcohols.126. The process of any of embodiments 122-124 wherein the compound(s)having a straight-chain alkyl group are fatty amides.127. The process of any of embodiments 122-124 wherein the compound(s)having a straight-chain alkyl group are fatty diacids or fatty diacidesters.128. The process of any of embodiments 122-124 wherein the compound(s)having a straight-chain alkyl group are fatty acids.129. The process of any of embodiments 122-124 wherein the compound(s)having a straight-chain alkyl group fatty acid esters.130. The process of embodiment 129 wherein the fatty acid esters aremethyl and/or ethyl esters.131. The process of embodiment 130 wherein the fermentation mediumincludes methanol and/or ethanol.132. The process of any of embodiments 122-131 wherein at least 60% byweight of the compound(s) having a straight-chain alkyl group have 8carbon atoms in the straight-chain alkyl group.133. The process of any of embodiments 122-131 wherein at least 80% byweight of the compound(s) having a straight-chain alkyl group have 8carbon atoms in the straight-chain alkyl group.134. The process of any of embodiments 122-131 wherein at least 90% byweight of the compound(s) having a straight-chain alkyl group have 8carbon atoms in the straight-chain alkyl group.135. The process of any of embodiments 122-131 wherein at least 60% byweight of the compound(s) having a straight-chain alkyl group have 10carbon atoms in the straight-chain alkyl group.136. The process of any of embodiments 122-131 wherein at least 80% byweight of the compound(s) having a straight-chain alkyl group have 10carbon atoms in the straight-chain alkyl group.137. The process of any of embodiments 122-131 wherein at least 90% byweight of the compound(s) having a straight-chain alkyl group have 8carbon atoms in the straight-chain alkyl group.138. The process of any of embodiments 122-131 wherein at least 95% byweight of the compound(s) having a straight-chain alkyl group have 8carbon atoms in the straight-chain alkyl group.139. The process of any of embodiments 122-138 wherein at least 0.05grams of the one or more compound(s) having a straight-chain alkyl groupare produced per liter of fermentation broth per hour.140. The process of any of embodiments 122-138 wherein at least 0.1grams of the one or more compound(s) having a straight-chain alkyl groupare produced per liter of fermentation broth per hour.141. The process of any of embodiments 122-138 wherein at least 0.25grams of the one or more compound(s) having a straight-chain alkyl groupare produced per liter of fermentation broth per hour.142. Use of a genetically modified cell of any of embodiments 95-121 toproduce one or more compounds having a straight-chain alkyl group,wherein at least 60% by weight of the one or more compounds has 6 to 10carbon atoms in the straight-chain alkyl group.145. Use of a genetically modified cell of any of embodiments 95-121 toproduce one or more compounds having a straight-chain alkyl group,wherein at least 80% by weight of the one or more compounds has 8 to 10carbon atoms in the straight-chain alkyl group.146. A cell that produces a 3-ketoacyl-CoA synthase of any ofembodiments 1-93.

1. A ketoacyl-CoA synthase having SEQ ID NO. 119 or being at least 80%identical to SEQ ID NO. 119, wherein the amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ IDNO. 119 is isoleucine, leucine, methionine or valine.
 2. Theketoacyl-CoA synthase of claim 1, comprising at least one of thefollowing features i) to xliii): i) an amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 18 of SEQ IDNO. 119 is alanine; ii) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 22 of SEQ ID NO. 119 ismethionine; iii) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 30 of SEQ ID NO. 119 is alanine; iv) anamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 38 of SEQ ID NO. 119 is valine; v) an amino acid residue ofthe ketoacyl-CoA synthase that aligns with amino acid residue 39 of SEQID NO. 119 is valine; vi) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 51 of SEQ ID NO. 119 isalanine, cysteine, aspartic acid, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan or tyrosine; vii) an amino acid residue ofthe ketoacyl-CoA synthase that aligns with amino acid residue 54 of SEQID NO. 119 is valine; viii) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 69 of SEQ ID NO. 119 isvaline; ix) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 83 of SEQ ID NO. 119 is asparagine; x) anamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 94 of SEQ ID NO. 119 is threonine, leucine, glutamic acid,or alanine; xi) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 111 of SEQ ID NO. 119 is cysteine; xii)an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 116 of SEQ ID NO. 119 is glycine; xiii) an an aminoacid residue of the ketoacyl-CoA synthase that aligns with amino acidresidue 127 of SEQ ID NO. 119 is a threonine; xiv) an amino acid residueof the ketoacyl-CoA synthase that aligns with amino acid residue 130 ofSEQ ID NO. 119 is glycine; xv) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 152 of SEQ ID NO. 119 iscysteine, leucine, methionine or threonine; xvi) an amino acid residueof the ketoacyl-CoA synthase that aligns with amino acid residue 154 ofSEQ ID NO. 119 is glycine; xvii) an an amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 178 of SEQ IDNO. 119 is leucine or threonine; xviii) an amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 190 of SEQ IDNO. 119 is phenylalanine Or tyrosine; xix) an amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 210 of SEQ IDNO. 119 is valine; xx) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 223 of SEQ ID NO. 119 ishistidine; xxi) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 231 of SEQ ID NO. 119 is isoleucine;xxii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 232 of SEQ ID NO. 119 is valine; xxiii) an aminoacid residue of the ketoacyl-CoA synthase that aligns with amino acidresidue 236 of SEQ ID NO. 119 is leucine or methionine; xxiv) an aminoacid residue of the ketoacyl-CoA synthase that aligns with amino acidresidue 241 of SEQ ID NO. 119 is proline; xxv) an amino acid residue ofthe ketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQID NO. 119 is alanine; xxvi) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 271 of SEQ ID NO. 119 isisoleucine; xxvii) an amino acid residue of the ketoacyl-CoA synthasethat aligns with amino acid residue 274 of SEQ ID NO. 119 is glutamicacid; xxviii) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 278 of SEQ ID NO. 119 is arginine,glutamic acid, aspartic acid, glutamine; xxix) an amino acid residue ofthe ketoacyl-CoA synthase that aligns with amino acid residue 280 of SEQID NO. 119 is isoleucine; xxx) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 282 of SEQ ID NO. 119 isglycine; xxxi) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 296 of SEQ ID NO. 119 is alanine; xxxii)an amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 302 of SEQ ID NO. 119 is threonine; xxxiii) an aminoacid residue of the ketoacyl-CoA synthase that aligns with amino acidresidue 312 of SEQ ID NO. 119 is aspartic acid; xxxiv) an amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue313 of SEQ ID NO. 119 is glutamic acid or methionine; xxxv) an aminoacid residue of the ketoacyl-CoA synthase that aligns with amino acidresidue 315 of SEQ ID NO. 119 is lysine; xxxvi) an amino acid residue ofthe ketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQID NO. 119 is alanine; xxxvii) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 322 of SEQ ID NO. 119 isglycine; xxxviii) an amino acid residue of the ketoacyl-CoA synthasethat aligns with amino acid residue 328 of SEQ ID NO. 119 is glycine;xxxix) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 329 of SEQ ID NO. 119 is glycine; xl) an aminoacid residue of the ketoacyl-CoA synthase that aligns with amino acidresidue 344 of SEQ ID NO. 119 is glutamic acid, aspartic acid orasparagine; xli) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 356 of SEQ ID NO. 119 is glycine orserine; xlii) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 368 of SEQ ID NO. 119 is arginine; andxliii) an amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 370 of SEQ ID NO. 119 is threonine.
 3. Theketoacyl-CoA synthase of claim 1 wherein the amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 184 of SEQ IDNO. 119 is isoleucine.
 4. The ketoacyl-CoA synthase of claim 1 furthercomprising at least one of the following features: the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue268 of SEQ ID NO. 119 is alanine; the amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 296 of SEQ IDNO. 119 is alanine; and the amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 328 of SEQ ID NO. 119 isglycine.
 5. The ketoacyl-CoA synthase of claim 1 wherein the amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue317 of SEQ ID NO. 119 is alanine.
 6. The ketoacyl-CoA synthase of claim2 wherein the amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 30 of SEQ ID NO. 119 is alanine.
 7. Theketoacyl-CoA synthase of claim 2 wherein the amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 368 of SEQ IDNO. 119 is arginine.
 8. The ketoacyl-CoA synthase of claim 1 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 51 of SEQ ID NO. 119 is alanine, cysteine, aspartic acid,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine.9. The ketoacyl-CoA synthase of claim 1 wherein the amino acid residueof the ketoacyl-CoA synthase that aligns with amino acid residue 51 ofSEQ ID NO. 119 is alanine.
 10. The 3-ketoacyl-CoA synthase of claim 1wherein the amino acid sequence is selected from the group consisting ofSEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ IDNO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQID NO. 23, SEQ 1D NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27,SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 37, SEQ IDNO. 38, SEQ ID NO. 39, SEQ ID NO. 92, SEQ ID NO. 93, any of SEQ ID NOs.121-157 and any one of SEQ ID NOs. 172-204.
 11. A ketoacyl-CoA synthasehaving SEQ ID NO. 110 or being at least 80% identical to SEQ ID NO. 110,comprising at least one of features i)-xiii): i) an amino acid residueof the ketoacyl-CoA synthase that aligns with amino acid residue 30 ofSEQ ID NO. 110 is alanine; ii) an amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 39 of SEQ ID NO. 110 isvaline; iii) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 69 of SEQ ID NO. 110 is valine; iv) anamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 111 of SEQ ID NO. 110 is cysteine; v) an amino acid residueof the ketoacyl-CoA synthase that aligns with amino acid residue 152 ofSEQ ID NO. 110 is cysteine, leucine, methionine or threonine; vi) anamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 178 of SEQ ID NO. 110 is leucine or threonine; vii) anamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 184 of SEQ ID NO. 110 is isoleucine, leucine, methionine orvaline; viii) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 268 of SEQ ID NO. 110 is alanine; ix) anamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 271 of SEQ ID NO. 110 is isoleucine; x) an amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue278 of SEQ ID NO. 110 is arginine, glutamic acid, aspartic acid, orglutamine; xi) an amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 296 of SEQ ID NO. 110 is alanine; xii) anamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 317 of SEQ ID NO. 110 is alanine; xiii) an amino acidresidue of the ketoacyl-CoA synthase that aligns with amino acid residue328 of SEQ ID NO. 110 is glycine.
 12. The ketoacyl-CoA synthase of claim11 wherein the amino acid residue of the ketoacyl-CoA synthase thataligns with amino acid residue 184 of SEQ ID NO. 110 is isoleucine,leucine, methionine or valine.
 13. The ketoacyl-CoA synthase of claim 11wherein the amino acid residue of the ketoacyl-CoA synthase that alignswith amino acid residue 184 of SEQ ID NO. 110 is isoleucine.
 14. Theketoacyl-CoA synthase of claim 11 wherein: the amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 268 of SEQ IDNO. 110 is alanine; the amino acid residue of the ketoacyl-CoA synthasethat aligns with amino acid residue 296 of SEQ ID NO. 110 is alanine;and the amino acid residue of the ketoacyl-CoA synthase that aligns withamino acid residue 328 of SEQ ID NO. 110 is glycine.
 15. Theketoacyl-CoA synthase of claim 11 wherein the amino acid residue of theketoacyl-CoA synthase that aligns with amino acid residue 317 of SEQ IDNO. 110 is alanine.
 16. The ketoacyl-CoA synthase claim 12 wherein theamino acid residue of the ketoacyl-CoA synthase that aligns with aminoacid residue 30 of SEQ ID NO. 110 is alanine.
 17. The ketoacyl-CoAsynthase of claim 12 wherein the amino acid residue of the ketoacyl-CoAsynthase that aligns with amino acid residue 368 of SEQ ID NO. 110 isarginine. 18.-26. (canceled)
 27. A 3-ketoacyl-CoA synthase gene encodingfor a 3-ketoacyl-CoA synthase of claim
 1. 28. A genetically modifiedcell comprising a 3-ketoacyl-CoA synthase gene of claim
 27. 29.-32.(canceled)